Anti-LAG-3 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

1. RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/730,249 (allowed), filed Oct. 11, 2017, which claims the benefit ofU.S. Provisional Application Nos.: 62/406,766, filed Oct. 11, 2016; and62/420,280, filed Nov. 10, 2016, each of which is incorporated byreference herein in its entirety.

2. FIELD

The instant disclosure relates to antibodies that specifically bind toLAG-3 (e.g., human LAG-3) and methods of using the same.

3. BACKGROUND

Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a type Imembrane protein in the immunoglobulin (Ig) superfamily that is composedof four extracellular Ig domains and a cytoplasmic domain containing aconserved repeated EP motif and a single conserved KIEELE motif (Triebelet al., (1990) J Exp Med, 171: 1393-405; Workman et al., (2002) JImmunol, 169: 5392-5). LAG-3 is expressed on activated effector Tlymphocytes (Teff), activated regulatory T lymphocytes (Treg), activatedB lymphocytes, a subset of resting natural killer (NK) cells, andresting plasmacytoid dendritic cells (PDC) (Huang et al., (2004)Immunity, 21: 503-13; Workman et al., (2009) J Immunol, 182: 1885-91;Kisielow et al., (2005) Eur J Immunol, 35: 2081-8; Baixeras et al.,(1992) J Exp Med, 176: 327-37; Workman et al., (2002) Eur J Immunol, 32:2255-63). Under conditions of persistent antigenic exposure, such as inchronic pathogenic infections or within the tumor microenvironment(TME), LAG-3 expression is sustained on T regulatory type 1 cells (Trl)and so-called exhausted antigen-specific T cells (Park et al., (2012)Cell Immunol, 278: 76-83; Gagliani et al., (2013) Nat Med, 19: 739-46;Blackburn et al., (2009) Nat Immunol, 10: 29-37).

LAG-3 functions to negatively regulate activated T cells. The ligand forLAG-3 is MHC class II, expressed on antigen presenting cells (APC) andactivated T cells (Roche and Furuta (2015) Nat Rev Immunol, 15: 203-16).The interaction between LAG-3 and its ligand inhibits proliferation andcytokine secretion of CD4+ and CD8+ Teff cells (Macon-Lemaitre andTriebel (2005) Immunology, 115: 170-8; Huard et al., (1997) Proc NatlAcad Sci USA, 94: 5744-9). LAG-3 in Tregs and PDCs contributes to thenegative regulation of T cell function (Huang et al., (2004) Immunity,21: 503-13; Workman et al., (2009) J Immunol, 182: 1885-91). Consistentwith its role in maintaining immune homeostasis, LAG-3 deficiencyinduced lethal myocarditis in mice also genetically deficient in PD-1(Okazaki et al., (2011) J Exp Med, 208: 395-407). Furthermore, in vivoblockade with a monoclonal antibody against mouse LAG-3 in combinationwith PD-1 blockade synergized to potentiate anti-tumor immunity insyngeneic mouse tumor models (Woo et al., (2012) Cancer Res, 72:917-27).

Given LAG-3's role in modulating immune responses, therapeutic agentsdesigned to antagonize LAG-3 signaling hold great promise for thetreatment of diseases that involve LAG-3-mediated immune suppression.

4. SUMMARY

The instant disclosure provides antibodies that specifically bind toLAG-3 (e.g., human LAG-3) and antagonize LAG-3 function, e.g.,LAG-3-mediated immune suppression. Also provided are pharmaceuticalcompositions comprising these antibodies, nucleic acids encoding theseantibodies, expression vectors and host cells for making theseantibodies, and methods of treating a subject using these antibodies.The antibodies disclosed herein are particularly useful for increasing Tcell activation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen) and/or decreasing Treg-mediated immunesuppression, and hence for treating cancer in a subject or treating orpreventing an infectious disease in a subject.

Accordingly, in one aspect, the instant disclosure provides an antibodyor isolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3 and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein:

-   -   (a) CDRH1 comprises the amino acid sequence of DX₁YX₂X₃ (SEQ ID        NO: 140), wherein        -   X₁ is T or N,        -   X₂ is I or M, and        -   X₃ is H, Y or D;    -   (b) CDRH2 comprises the amino acid sequence of        X₁IDPANX₂X₃X₄X₅X₆X₇PX₈X₉QX₁₀ (SEQ ID NO: 142), wherein        -   X₁ is E, R, S, or K,        -   X₂ is D or G,        -   X₃ is N or H,        -   X₄ is T or S,        -   X₅ is K or H,        -   X₆ is Y or F,        -   X₇ is D or A,        -   X₅ is K or R,        -   X₉ is F or L, and        -   X₁₀ is G or D;    -   (c) CDRH3 comprises the amino acid sequence of YX₁X₂X₃YX₄VGGX₅DY        (SEQ ID NO: 144), wherein        -   X₁ is Y, F, or S,        -   X₂ is Y or D,        -   X₃ is K or R,        -   X₄ is D or E, and        -   X₅ is F or C;    -   (d) CDRL1 comprises the amino acid sequence of SVSSX₁ISSSX₂LX₃        (SEQ ID NO: 147), wherein        -   X₁ is S or G,        -   X₂ is N or T, and        -   X₃ is H or Y;    -   (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID        NO: 104); and    -   (f) CDRL3 comprises the amino acid sequence of QQWX₁X₂YPX₃T (SEQ        ID NO: 149), wherein        -   X₁ is S, N, or R,        -   X₂ is S, T or R, and        -   X₃ is F, L, H, or W.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain variable region comprising complementaritydetermining regions CDRH1, CDRH2 and CDRH3 and a light chain variableregion comprising complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein:

-   -   (a) CDRH1 comprises the amino acid sequence of DX₁YX₂X₃ (SEQ ID        NO: 140), wherein        -   X₁ is T or N,        -   X₂ is I or M, and        -   X₃ is H, Y or D;    -   (b) CDRH2 comprises the amino acid sequence of        X₁IDPANX₂X₃X₄X₅X₆X₇PX₈X₉QX₁₀ (SEQ ID NO: 142), wherein        -   X₁ is E, R, S, or K,        -   X₂ is D or G,        -   X₃ is N or H,        -   X₄ is T or S,        -   X₅ is K or H,        -   X₆ is Y or F,        -   X₇ is D or A,        -   X₈ is K or R,        -   X₉ is F or L, and        -   X₁₀ is G or D;    -   (c) CDRH3 comprises the amino acid sequence of YX₁X₂X₃YX₄VGGX₅DY        (SEQ ID NO: 144), wherein        -   X₁ is Y, F, or 5,        -   X₂ is Y or D,        -   X₃ is K or R,        -   X₄ is D or E, and        -   X₅ is F or C;    -   (d) CDRL1 comprises the amino acid sequence of SVSSX₁ISSSX₂LX₃        (SEQ ID NO: 147), wherein        -   X₁ is S or G,        -   X₂ is N or T, and        -   X₃ is H or Y;    -   (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID        NO: 104); and    -   (f) CDRL3 comprises the amino acid sequence of QQWX₁X₂YPX₃T (SEQ        ID NO: 149), wherein        -   X₁ is S, N, or R,        -   X₂ is S, T or R, and        -   X₃ is F, L, H, or W.

In certain embodiments, CDRH1 comprises the amino acid sequence ofDX₁YX₂X₃ (SEQ ID NO: 141), wherein: X₁ is T or N; X₂ is I or M; and X₃is H or Y. In certain embodiments, CDRH2 comprises the amino acidsequence of X₁IDPANX₂X₃X₄KX₅X₆PX₇FQX₈ (SEQ ID NO: 143), wherein: X₁ isE, R, or S; X₂ is D or G; X₃ is N or H; X₄ is T or S; X₅ is Y or F; X₆is D or A; X₇ is K or R; and X₈ is G or D. In certain embodiments, CDRH3comprises the amino acid sequence of YX₁X₂X₃YDVGGX₄DY (SEQ ID NO: 145),wherein: X₁ is Y, F, or S; X₂ is Y or D; X₃ is K or R; and X₄ is F or C.In certain embodiments, CDRH3 comprises the amino acid sequence ofYYYX₁YX₂VGGFDY (SEQ ID NO: 146), wherein: X₁ is K or R; and X₂ is D orE. In certain embodiments, CDRL1 comprises the amino acid sequence ofSVSSSISSSNLX₁ (SEQ ID NO: 148), wherein: X₁ is H or Y. In certainembodiments, CDRL3 comprises the amino acid sequence of QQWX₁SYPX₂T (SEQID NO: 150), wherein: X₁ is S, N, or R; and X₂ is F, L, or H.

In certain embodiments:

-   -   (a) CDRH1 comprises the amino acid sequence of DTYIH (SEQ ID NO:        79);    -   (b) CDRH2 comprises the amino acid sequence of EIDPANDNTKYDPKFQG        (SEQ ID NO: 90);    -   (c) CDRH3 comprises the amino acid sequence of YYYX₁YX₂VGGFDY        (SEQ ID NO: 146), wherein: X₁ is K or R; and X₂ is D or E;    -   (d) CDRL1 comprises the amino acid sequence of SVSSSISSSNLH (SEQ        ID NO: 100);    -   (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID        NO: 104); and    -   (f) CDRL3 comprises the amino acid sequence of QQWSSYPFT (SEQ ID        NO: 105).

In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acidsequences set forth in SEQ ID NOs: 79, 90, and 98, respectively. Incertain embodiments, the heavy chain variable region comprises an aminoacid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certainembodiments, the heavy chain variable region comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 65 or 220. In certain embodiments, the heavy chain variableregion comprises the amino acid sequence of SEQ ID NO: 220. In certainembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 65. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 220. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 226. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 169.

In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acidsequences set forth in SEQ ID NOs: 100, 104, and 105, respectively. Incertain embodiments, the light chain variable region comprises an aminoacid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certainembodiments, the light chain variable region comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 73 or 221. In certain embodiments, the light chain variableregion comprises the amino acid sequence of SEQ ID NO: 221. In certainembodiments, the light chain variable region comprises the amino acidsequence of SEQ ID NO: 73. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:228. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 187.

In certain embodiments, CDRH1 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 78-82. In certain embodiments,CDRH2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 83-93. In certain embodiments, CDRH3 comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:94-99. In certain embodiments, CDRL1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 100-103. In certainembodiments, CDRL3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 105-112.

In certain embodiments, CDRH1, CDRH2 and CDRH3 comprise the CDRH1, CDRH2and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs:78, 83, and 94; 78, 85, and 95; 78, 86, and 96; 78, 86, and 97; 78, 91,and 94; 78, 92, and 96; 79, 84, and 95; 79, 88, and 95; 79, 89, and 95;79, 90, and 95; 79, 90, and 98; 79, 90, and 99; 80, 85, and 96; 81, 87,and 96; or, 82, 93, and 95.

In certain embodiments, CDRL1, CDRL2 and CDRL3 comprise the CDRL1, CDRL2and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs:100, 104, and 105; 100, 104, and 106; 100, 104, and 107; 100, 104, and109; 100, 104, and 110; 101, 104, and 108; 102, 104, and 105; 102, 104,and 112; or, 103, 104, and 111.

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the amino acid sequences set forth in SEQ ID NOs: 78, 83, 94,100, 104, and 105; 78, 85, 95, 100, 104, and 105; 78, 86, 96, 100, 104,and 105; 78, 86, 96, 100, 104, and 109; 78, 86, 96, 100, 104, and 110;78, 86, 96, 101, 104, and 108; 78, 86, 96, 103, 104, and 111; 78, 86,97, 102, 104, and 112; 78, 91, 94, 100, 104, and 107; 78, 92, 96, 100,104, and 105; 78, 92, 96, 100, 104, and 109; 79, 84, 95, 100, 104, and105; 79, 84, 95, 100, 104, and 106; 79, 84, 95, 102, 104, and 105; 79,88, 95, 100, 104, and 105; 79, 89, 95, 100, 104, and 105; 79, 90, 95,100, 104, and 105; 79, 90, 98, 100, 104, and 105; 79, 90, 99, 100, 104,and 105; 80, 85, 96, 100, 104, and 105; 81, 87, 96, 100, 104, and 105;81, 87, 96, 100, 104, and 107; or, 82, 93, 95, 100, 104, and 105,respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90,95, 100, 104, and 105, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 79, 90, 95, 100, 104, and 105,respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90,98, 100, 104, and 105, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 79, 90, 98, 100, 104, and 105,respectively.

In certain embodiments, the heavy chain variable region comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. Incertain embodiments, the heavy chain variable region comprises an aminoacid sequence that is at least 98% identical to the amino acid sequenceof SEQ ID NO: 65 or 220. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 220. Incertain embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 65. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:226. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 169.

In certain embodiments, the light chain variable region comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. Incertain embodiments, the light chain variable region comprises an aminoacid sequence that is at least 98% identical to the amino acid sequenceof SEQ ID NO: 73 or 221. In certain embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 221. Incertain embodiments, the light chain variable region comprises the aminoacid sequence of SEQ ID NO: 73. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:228. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 228. In certainembodiments, the antibody comprises a light chain comprising the aminoacid sequence of SEQ ID NO: 187.

In certain embodiments, the heavy chain variable region comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220, andthe light chain variable region comprises an amino acid sequence that isat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, theheavy chain variable region comprises an amino acid sequence that is atleast 98% identical to the amino acid sequence of SEQ ID NO: 65 or 220,and the light chain variable region comprises an amino acid sequencethat is at least 98% identical to the amino acid sequence of SEQ ID NO:73 or 221. In certain embodiments, the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 220. In certainembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 65. In certain embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 73 or221. In certain embodiments, the light chain variable region comprisesthe amino acid sequence of SEQ ID NO: 73.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human LAG-3, comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 65 or220. In certain embodiments, the antibody comprises a light chainvariable region comprising an amino acid sequence that is at least 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 73 or 221. In certain embodiments, the antibodycomprises a light chain variable region comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 73 or 221.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human LAG-3, comprising a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 73 or221. In certain embodiments, the antibody comprises a heavy chainvariable region comprising an amino acid sequence that is at least 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 65 or 220. In certain embodiments, the antibodycomprises a heavy chain variable region comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 65 or 220.

In certain embodiments, the antibody comprises a heavy chain variableregion comprising the framework regions of the heavy chain variableregion sequence of SEQ ID NO: 151 or 222. In certain embodiments, theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 151 or 222. In certain embodiments, theantibody comprises a heavy chain variable region comprising theframework regions of the heavy chain variable region sequence of SEQ IDNO: 218 or 223. In certain embodiments, the antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:218 or 223. In certain embodiments, the antibody comprises a heavy chainvariable region comprising an amino acid sequence which is at least 75%,80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 56-72 and 220. In certainembodiments, the heavy chain variable region comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 56-72 and220. In certain embodiments, the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO: 220. In certain embodiments, theheavy chain variable region comprises the amino acid sequence of SEQ IDNO: 65. In certain embodiments, the antibody comprises a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 168-186 and 225-227. In certain embodiments, the antibodycomprises a heavy chain variable region having human derived frameworkregions. In certain embodiments, the antibody comprises a heavy chainvariable framework region that is or is derived from an amino acidsequence encoded by a human gene, wherein said amino acid sequence isselected from the group consisting of IGHV1-46*01 (SEQ ID NO: 153),IGHV1-69-2*01 (SEQ ID NO: 154), IGHV1-3*01 (SEQ ID NO: 155), IGHV1-24*01(SEQ ID NO: 156), IGHV1-2*01 (SEQ ID NO: 157), IGHV1-45*01 (SEQ ID NO:158), and IGHV1-18*01 (SEQ ID NO: 159). In certain embodiments, theantibody comprises a heavy chain variable framework region that isderived from the amino acid sequence IGHV1-46*01 (SEQ ID NO: 153),wherein at least one amino acid in the amino acid sequence IGHV1-46*01(SEQ ID NO: 153) is substituted with an amino acid in an analogousposition in a corresponding non-human heavy chain variable frameworkregion. In certain embodiments, the amino acid substitution is at anamino acid position selected from the group consisting of 4, 5, 12, 23,27, 28, 29, 30, 48, 69, 71, 75, 76, 80, 81, and 94, wherein the aminoacid position is indicated according to the Kabat numbering system. Incertain embodiments, the amino acid substitution is selected from thegroup consisting of 4M, 5K, 12V, 23T, 27F, 28N, 291, 30K, 48I, 69I, 71A,75S, 76N, 80L, 81Q, and 94T, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system. Incertain embodiments, the amino acid substitution is at an amino acidposition selected from the group consisting of 4, 27, 28, 29, 30, 69,71, and 94, wherein the amino acid position is indicated according tothe Kabat numbering system. In certain embodiments, the amino acidsubstitution is selected from the group consisting of 4M, 27F, 28N, 291,30K, 691, 71A, and 94T, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system.

In certain embodiments, the antibody comprises a light chain variableregion comprising the framework regions of the light chain variableregion sequence of SEQ ID NO: 152 or 224. In certain embodiments, theantibody comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 152 or 224. In certain embodiments, theantibody comprises a light chain variable region comprising an aminoacid sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 73-77 and 221. In certain embodiments, the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 73-77 and 221. In certain embodiments, thelight chain variable region comprises the amino acid sequence of SEQ IDNO: 221. In certain embodiments, the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 73. In certainembodiments, the antibody comprises a light chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 187-191.In certain embodiments, the antibody comprises a light chain variableregion having human derived framework regions. In certain embodiments,the antibody comprises a light chain variable framework region that isor is derived from an amino acid sequence encoded by a human gene,wherein said amino acid sequence is selected from the group consistingof IGKV3-20*01 (SEQ ID NO: 160), IGKV3D-15*01 (SEQ ID NO: 161),IGKV3-15*01 (SEQ ID NO: 161), IGKV3D-20*01 (SEQ ID NO: 162), IGKV3D-7*01(SEQ ID NO: 163), IGKV1-9*01 (SEQ ID NO: 164), and IGKV3-11*01 (SEQ IDNO: 165). In certain embodiments, the antibody comprises a light chainvariable framework region that is from the amino acid sequenceIGKV3-20*01 (SEQ ID NO: 160). In certain embodiments, the antibodycomprises a light chain variable framework region that is derived fromthe amino acid sequence IGKV3-20*01 (SEQ ID NO: 160), wherein at leastone amino acid in the amino acid sequence IGKV3-20*01 (SEQ ID NO: 160)is substituted with an amino acid in an analogous position in acorresponding non-human light chain variable framework region. Incertain embodiments, the amino acid substitution is at an amino acidposition selected from the group consisting of 3, 22, 36, 43, 47, 58,70, and 71, wherein the amino acid position is indicated according tothe Kabat numbering system. In certain embodiments, the amino acidsubstitution is selected from the group consisting of 3L, 22T, 36F, 43S,47W, 58V, 70S, and 71Y, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:56-72 and 220. In certain embodiments, the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 220. In certainembodiments, the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 65. In certain embodiments, the antibodycomprises a heavy chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 168-186 and 225-227. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 225. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:168. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 226. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 169. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:227. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 170.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 56-72 and220. In certain embodiments, the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO: 220. In certain embodiments, theheavy chain variable region comprises the amino acid sequence of SEQ IDNO: 65. In certain embodiments, the antibody comprises a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 168-186 and 225-227. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:225. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 168. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 226. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:169. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 227. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 170.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:73-77 and 221. In certain embodiments, the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 221. In certainembodiments, the light chain variable region comprises the amino acidsequence of SEQ ID NO: 73. In certain embodiments, the antibodycomprises a light chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 187-191, and 228. In certainembodiments, the antibody comprises a light chain comprising the aminoacid sequence of SEQ ID NO: 228. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 73-77 and221. In certain embodiments, the light chain variable region comprisesthe amino acid sequence of SEQ ID NO: 221. In certain embodiments, thelight chain variable region comprises the amino acid sequence of SEQ IDNO: 73. In certain embodiments, the antibody comprises a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 187-191, and 228. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:228. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region and thelight chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68and 73; 69 and 73; 70 and 73; 71 and 73; or 72 and 73. In certainembodiments, the heavy chain variable region and the light chainvariable region, respectively, comprise the amino acid sequences setforth in SEQ ID NOs: 65 and 73. In certain embodiments, the heavy chainvariable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 220 and 73;65 and 221; or 220 and 221.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region and a lightchain variable region, wherein the amino acid sequences of the heavychain variable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 56 and 73;56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73; or 72 and73. In certain embodiments, the amino acid sequences of the heavy chainvariable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 65 and 73.In certain embodiments, the amino acid sequences of the heavy chainvariable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 220 and 73;65 and 221; or 220 and 221.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region and the light chainvariable region, respectively, comprise the amino acid sequences setforth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73;70 and 73; 71 and 73; or 72 and 73. In certain embodiments, the heavychain variable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 65 and 73. Incertain embodiments, the heavy chain variable region and the light chainvariable region, respectively, comprise the amino acid sequences setforth in SEQ ID NOs: 220 and 73; 65 and 221; or 220 and 221.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the amino acid sequences of the heavy chain variableregion and the light chain variable region, respectively, consist of theamino acid sequences set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73; or 72 and 73. Incertain embodiments, the amino acid sequences of the heavy chainvariable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 65 and 73.In certain embodiments, the amino acid sequences of the heavy chainvariable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 220 and 73;65 and 221; or 220 and 221.

In certain embodiments of any one of the foregoing aspects whereapplicable, the X in SEQ ID NO: 220 is Q. In certain embodiments of anyone of the foregoing aspects where applicable, the X in SEQ ID NO: 220is pyroglutamate. In certain embodiments of any one of the foregoingaspects where applicable, the X in SEQ ID NO: 221 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 221 is pyroglutamate. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 220 is Q,and the X in SEQ ID NO: 221 is E. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 220 is Q,and the X in SEQ ID NO: 221 is pyroglutamate. In certain embodiments ofany one of the foregoing aspects where applicable, the X in SEQ ID NO:220 is pyroglutamate, and the X in SEQ ID NO: 221 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 220 is pyroglutamate, and the X in SEQ ID NO: 221 ispyroglutamate.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 168 or 225, and a light chain comprising theamino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 168, and a light chain comprising the amino acidsequence of SEQ ID NO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 168 or 225, and a light chain comprising the amino acid sequence ofSEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 168, and a light chain comprising the amino acid sequence of SEQ IDNO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 169 or 226, and a light chain comprising theamino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 169, and a light chain comprising the amino acidsequence of SEQ ID NO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 169 or 226, and a light chain comprising the amino acid sequence ofSEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 169, and a light chain comprising the amino acid sequence of SEQ IDNO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 170 or 227, and a light chain comprising theamino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 170, and a light chain comprising the amino acidsequence of SEQ ID NO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 170 or 227, and a light chain comprising the amino acid sequence ofSEQ ID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 170, and a light chain comprising the amino acid sequence of SEQ IDNO: 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequence of the heavy chain consists of the amino acidsequence set forth in SEQ ID NO: 168 or 225, and the amino acid sequenceof the light chain consists of the amino acid sequence set forth in SEQID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequences of the heavy chain and the light chain,respectively, consist of the amino acid sequences set forth in SEQ IDNOs: 168 and 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequence of the heavy chain consists of the amino acid sequence setforth in SEQ ID NO: 168 or 225, and the amino acid sequence of the lightchain consists of the amino acid sequence set forth in SEQ ID NO: 187 or228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequences of the heavy chain and the light chain, respectively, consistof the amino acid sequences set forth in SEQ ID NOs: 168 and 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequence of the heavy chain consists of the amino acidsequence set forth in SEQ ID NO: 169 or 226, and the amino acid sequenceof the light chain consists of the amino acid sequence set forth in SEQID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequences of the heavy chain and the light chain,respectively, consist of the amino acid sequences set forth in SEQ IDNOs: 169 and 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequence of the heavy chain consists of the amino acid sequence setforth in SEQ ID NO: 169 or 226, and the amino acid sequence of the lightchain consists of the amino acid sequence set forth in SEQ ID NO: 187 or228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequences of the heavy chain and the light chain, respectively, consistof the amino acid sequences set forth in SEQ ID NOs: 169 and 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequence of the heavy chain consists of the amino acidsequence set forth in SEQ ID NO: 170 or 227, and the amino acid sequenceof the light chain consists of the amino acid sequence set forth in SEQID NO: 187 or 228.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain and a light chain, whereinthe amino acid sequences of the heavy chain and the light chain,respectively, consist of the amino acid sequences set forth in SEQ IDNOs: 170 and 187.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequence of the heavy chain consists of the amino acid sequence setforth in SEQ ID NO: 170 or 227, and the amino acid sequence of the lightchain consists of the amino acid sequence set forth in SEQ ID NO: 187 or228.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human LAG-3, the antibodycomprising a heavy chain and a light chain, wherein the amino acidsequences of the heavy chain and the light chain, respectively, consistof the amino acid sequences set forth in SEQ ID NOs: 170 and 187.

In certain embodiments of any one of the foregoing aspects whereapplicable, the X in SEQ ID NO: 225 is Q. In certain embodiments of anyone of the foregoing aspects where applicable, the X in SEQ ID NO: 225is pyroglutamate. In certain embodiments of any one of the foregoingaspects where applicable, the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 225 is Q,and the X in SEQ ID NO: 228 is E. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 225 is Q,and the X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments ofany one of the foregoing aspects where applicable, the X in SEQ ID NO:225 is pyroglutamate, and the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 225 is pyroglutamate, and the X in SEQ ID NO: 228 ispyroglutamate.

In certain embodiments of any one of the foregoing aspects whereapplicable, the X in SEQ ID NO: 226 is Q. In certain embodiments of anyone of the foregoing aspects where applicable, the X in SEQ ID NO: 226is pyroglutamate. In certain embodiments of any one of the foregoingaspects where applicable, the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 226 is Q,and the X in SEQ ID NO: 228 is E. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 226 is Q,and the X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments ofany one of the foregoing aspects where applicable, the X in SEQ ID NO:226 is pyroglutamate, and the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 226 is pyroglutamate, and the X in SEQ ID NO: 228 ispyroglutamate.

In certain embodiments of any one of the foregoing aspects whereapplicable, the X in SEQ ID NO: 227 is Q. In certain embodiments of anyone of the foregoing aspects where applicable, the X in SEQ ID NO: 227is pyroglutamate. In certain embodiments of any one of the foregoingaspects where applicable, the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 227 is Q,and the X in SEQ ID NO: 228 is E. In certain embodiments of any one ofthe foregoing aspects where applicable, the X in SEQ ID NO: 227 is Q,and the X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments ofany one of the foregoing aspects where applicable, the X in SEQ ID NO:227 is pyroglutamate, and the X in SEQ ID NO: 228 is E. In certainembodiments of any one of the foregoing aspects where applicable, the Xin SEQ ID NO: 227 is pyroglutamate, and the X in SEQ ID NO: 228 ispyroglutamate.

In certain embodiments, the antibody comprises a heavy chain constantregion selected from the group consisting of human IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂. In certain embodiments, the heavy chain constantregion is IgG₁. In certain embodiments, the amino acid sequence of IgG₁comprises a N297A mutation, numbered according to the EU numberingsystem. In certain embodiments, the antibody comprises a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 194. Incertain embodiments, the amino acid sequence of IgG₁ comprises a N297Qmutation, numbered according to the EU numbering system. In certainembodiments, the IgG₁ is afucosylated IgG₁. In certain embodiments, theheavy chain constant region is IgG₄. In certain embodiments, the aminoacid sequence of IgG₄ comprises a S228P mutation, numbered according tothe EU numbering system. In certain embodiments, the antibody comprisesa heavy chain constant region comprising the amino acid sequence of SEQID NO: 196.

In certain embodiments, the antibody comprises a light chain constantregion selected from the group consisting of human IgGκ and IgGλ. Incertain embodiments, the light chain constant region is IgGκ. In certainembodiments, the antibody comprises a light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 198. In certainembodiments, the antibody comprises a light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 219. In certainembodiments, the light chain constant region is IgGλ.

In another aspect, the instant disclosure provides an antibody orisolated antibody that cross-competes for binding to human LAG-3 with anantibody as disclosed herein. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human LAG-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 15 and 16, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human LAG-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 65 and 73; 220 and 73; 65 and 221; or 220 and 221, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds to the same epitope of human LAG-3 as anantibody disclosed herein. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human LAG-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 15 and 16, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human LAG-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 65 and 73; 220 and 73; 65 and 221; or 220 and 221, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds, e.g., specifically binds, to an epitope ofhuman LAG-3. In certain embodiments, the antibody binds to an epitopelocated within a region of human LAG-3 consisting of the amino acidsequence of SEQ ID NO: 216. In certain embodiments, the antibody bindsto an epitope located within a region of human LAG-3 consisting of theamino acid sequence of SEQ ID NO: 215. In certain embodiments, theantibody binds to an epitope located within a region of human LAG-3consisting of the amino acid sequence of SEQ ID NO: 214. In certainembodiments, the antibody binds to an epitope located within a region ofhuman LAG-3 consisting of the amino acid sequence of SEQ ID NO: 213. Incertain embodiments, the antibody binds to an epitope located within aregion of human LAG-3 consisting of the amino acid sequence of SEQ IDNO: 212. In certain embodiments, the antibody binds to an epitopelocated within a region of human LAG-3 consisting of the amino acidsequence of SEQ ID NO: 211.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanLAG-3 as any antibody of the present invention. In certain embodiments,the antibody binds to an epitope located within a region of human LAG-3consisting of the amino acid sequence of SEQ ID NO: 216. In certainembodiments, the antibody binds to an epitope located within a region ofhuman LAG-3 consisting of the amino acid sequence of SEQ ID NO: 215. Incertain embodiments, the antibody binds to an epitope located within aregion of human LAG-3 consisting of the amino acid sequence of SEQ IDNO: 214. In certain embodiments, the antibody binds to an epitopelocated within a region of human LAG-3 consisting of the amino acidsequence of SEQ ID NO: 213. In certain embodiments, the antibody bindsto an epitope located within a region of human LAG-3 consisting of theamino acid sequence of SEQ ID NO: 212. In certain embodiments, theantibody binds to an epitope located within a region of human LAG-3consisting of the amino acid sequence of SEQ ID NO: 211.

In another aspect, the instant disclosure provides an antibody that,when bound to a human LAG-3 protein or fragment thereof comprising theamino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuteriumexchange in a region consisting of the amino acid sequence set forth inSEQ ID NO: 216 relative to hydrogen/deuterium exchange in the regionconsisting of the amino acid sequence set forth in SEQ ID NO: 216 in theabsence of the antibody, as determined by a hydrogen/deuterium assay. Inanother aspect, the instant disclosure provides an antibody that, whenbound to a human LAG-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:215 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 215 in the absence ofthe antibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human LAG-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 214relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 214 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human LAG-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 213relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 213 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human LAG-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 212relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 212 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human LAG-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 211relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 211 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In someembodiments, the reduction in hydrogen/deuterium exchange is measuredusing hydrogen-deuterium exchange (HDX), for example as described hereinin the examples.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanLAG-3 as any antibody of the present invention. In certain embodiments,the antibody, when bound to a human LAG-3 protein or fragment thereofcomprising the amino acid sequence of SEQ ID NO: 217, reduceshydrogen/deuterium exchange in a region consisting of the amino acidsequence set forth in SEQ ID NO: 216 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 216 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human LAG-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:215 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 215 in the absence ofthe antibody, as determined by a hydrogen/deuterium assay. In certainembodiments, the antibody, when bound to a human LAG-3 protein orfragment thereof comprising the amino acid sequence of SEQ ID NO: 217,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 214 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 214 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human LAG-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:213 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 213 in the absence ofthe antibody, as determined by a hydrogen/deuterium assay. In certainembodiments, the antibody, when bound to a human LAG-3 protein orfragment thereof comprising the amino acid sequence of SEQ ID NO: 217,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 212 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 212 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human LAG-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:211 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 211 in the absence ofthe antibody, as determined by a hydrogen/deuterium assay. In someembodiments, the reduction in hydrogen/deuterium exchange is measuredusing hydrogen-deuterium exchange (HDX), for example as described hereinin the examples.

In certain embodiments, the antibody is a humanized antibody. In certainembodiments, the antibody is a murine antibody. In certain embodiments,the antibody is a chimeric antibody. In certain embodiments, theantibody is antagonistic to human LAG-3. In certain embodiments, theantibody deactivates, reduces, or inhibits an activity of human LAG-3.In certain embodiments, the antibody inhibits binding of human LAG-3 toMEW class II. In certain embodiments, the antibody induces IL-2production by peripheral blood mononuclear cells (PBMCs) stimulated withstaphylococcal enterotoxin A (SEA). In certain embodiments, the antibodyinduces TNFα production by tumor infiltrating lymphocytes (TILs)stimulated with anti-CD3 and anti-CD28 antibodies.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a cytotoxic agent.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a cytostatic agent.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a toxin.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a radionuclide.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a detectable label.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein, wherein the N-terminal amino acidresidue of the heavy chain variable region is pyroglutamate (e.g., as aresult of post-translational cyclization of the free amino group of theN-terminal E or Q residue of the heavy chain variable region). Inanother aspect, the instant disclosure provides an antibody or isolatedantibody as disclosed herein, wherein the N-terminal amino acid residueof the heavy chain is pyroglutamate (e.g., as a result ofpost-translational cyclization of the free amino group of the N-terminalE or Q residue of the heavy chain).

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein, wherein the N-terminal amino acidresidue of the light chain variable region is pyroglutamate (e.g., as aresult of post-translational cyclization of the free amino group of theN-terminal E or Q residue the light chain variable region). In anotheraspect, the instant disclosure provides an antibody or isolated antibodyas disclosed herein, wherein the N-terminal amino acid residue of thelight chain is pyroglutamate (e.g., as a result of post-translationalcyclization of the free amino group of the N-terminal E or Q residue thelight chain).

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein, in which the heavy chain isaglycosylated.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising an antibody as disclosed herein and apharmaceutically acceptable carrier or excipient.

In another aspect, the instant disclosure provides a polynucleotideisolated polynucleotide encoding a heavy and/or light chain of anantibody as disclosed herein. In another aspect, the instant disclosureprovides a vector comprising the polynucleotide. In another aspect, theinstant disclosure provides a recombinant host cell comprising thepolynucleotide. In another aspect, the instant disclosure provides arecombinant host cell comprising the vector. In another aspect, theinstant disclosure provides a method of producing an antibody asdisclosed herein, the method comprising culturing the host cell so thatthe polynucleotide is expressed and the antibody is produced. In oneembodiment, the method is an in vitro method.

In one embodiment, the present invention relates to an antibody of theinvention, or a pharmaceutical composition of the invention, or apolynucleotide of the invention, or a vector of the invention, or arecombinant host cell of the invention for use as a medicament.

In one embodiment, the present invention relates to an antibody of theinvention, or a pharmaceutical composition of the invention, or apolynucleotide of the invention, or a vector of the invention, or arecombinant host cell of the invention for use as a diagnostic.

In another aspect, the instant disclosure provides a method ofincreasing T cell activation in response to an antigen in a subject, themethod comprising administering to the subject an effective amount of anantibody or pharmaceutical composition as disclosed herein. In anotheraspect, the instant disclosure provides a method of treating cancer in asubject, the method comprising administering to the subject an effectiveamount of an antibody or pharmaceutical composition as disclosed herein.In certain embodiments of the foregoing methods, the antibody orpharmaceutical composition is administered subcutaneously. In certainembodiments of the foregoing methods, the antibody or pharmaceuticalcomposition is administered intravenously. In certain embodiments of theforegoing methods, the antibody or pharmaceutical composition isadministered intratumorally. In certain embodiments of the foregoingmethods, the antibody or pharmaceutical composition is delivered to atumor draining lymph node. In certain embodiments of the foregoingmethods, the antibody or pharmaceutical composition is administeredintra-arterially. In certain embodiments of the foregoing methods, theantibody or pharmaceutical composition is administered intranasally.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen in a subject comprisingadministering to the subject an effective amount of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the invention.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer in a subject comprising administering to the subjectan effective amount of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition of the invention.

In one embodiment of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition for use of the presentinvention, the antibody, polynucleotide, vector, recombinant host cell,and/or pharmaceutical composition is administered subcutaneously orintravenously. In one embodiment of an antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition for use of thepresent invention, the antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition is administeredintratumorally or intra-arterially. In one embodiment of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition for use of the present invention, the antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition is administered intranasally.

In certain embodiments, the foregoing methods further compriseadministering an additional therapeutic agent to the subject. Therefore,in one embodiment of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition for use in a method of thepresent invention, the method further comprises administering anadditional therapeutic agent to the subject.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent for use as a medicament.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent for use in a method for the treatment of cancer.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent.

In certain embodiments, the additional therapeutic agent is achemotherapeutic. In certain embodiments, the additional therapeuticagent is a radiotherapeutic.

In certain embodiments, the additional therapeutic agent is a checkpointtargeting agent. In certain embodiments, the checkpoint targeting agentis selected from the group consisting of an antagonist anti-PD-1antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1antibody, an agonist anti-GITR antibody, an agonist anti-OX40 antibody,an antagonist anti-TIGIT antibody, an agonist anti-CD137 antibody, anantagonist anti-VISTA antibody, an antagonist anti-CD73 antibody, and anantagonist anti-CD96 antibody. In certain embodiments, the additionaltherapeutic agent is an anti-PD-1 antibody. In certain embodiments, theanti-PD-1 antibody is pembrolizumab. In certain embodiments, theanti-PD-1 antibody is nivolumab. In certain embodiments, the additionaltherapeutic agent is an anti-PD-L1 antibody. In certain embodiments, theadditional therapeutic agent is an anti-CTLA-4 antibody.

In certain embodiments, the additional therapeutic agent comprises asmall molecule. In certain embodiments, the additional therapeutic agentis a small molecule inhibitor of the PD-1 pathway. In certainembodiments, the additional therapeutic agent is a small moleculeinhibitor of PD-1 or PD-L1.

In certain embodiments, the additional therapeutic agent is an inhibitorof indoleamine-2,3-dioxygenase (IDO). In certain embodiments, theinhibitor is selected from the group consisting of epacadostat,BMS-986205 (also known as F001287, see Example 19 of WO2016/073770,which is incorporated by reference herein in its entirety), indoximod,and NLG919. In certain embodiments, the inhibitor is epacadostat. Incertain embodiments, the inhibitor is BMS-986205. In certainembodiments, the inhibitor is indoximod. In certain embodiments, theinhibitor is NLG919.

In certain embodiments, the additional therapeutic agent is an inhibitorof ARG, LSD1, CD112, CD112R, or VEGF. In certain embodiments, theadditional therapeutic agent is a Stimulator of Interferon Genes (STING)agonist. In certain embodiments, the additional therapeutic agent is aCD80-Fc protein.

In certain embodiments, the additional therapeutic agent is a vaccine.In certain embodiments, the vaccine comprises a heat shock proteinpeptide complex (HSPPC) comprising a heat shock protein complexed withan antigenic peptide. In certain embodiments, the heat shock protein ishsc70 and is complexed with a tumor-associated antigenic peptide. Incertain embodiments, the heat shock protein is gp96 protein and iscomplexed with a tumor-associated antigenic peptide, wherein the HSPPCis derived from a tumor obtained from a subject. In certain embodiments,the heat shock protein is gp96 protein and is complexed with atumor-associated antigenic peptide, wherein the HSPPC is derived from atumor obtained from a subject. In certain embodiments, the additionaltherapeutic agent comprises a TCR. In certain embodiments, theadditional therapeutic agent is a soluble TCR. In certain embodiments,the additional therapeutic agent is a cell expressing a TCR. In certainembodiments, the additional therapeutic agent is a cell expressing achimeric antigen receptor. In certain embodiments, the additionaltherapeutic agent is an antibody that specifically binds to apeptide-MHC complex. In certain embodiments, the additional therapeuticagent is an adjuvant. In one aspect, the present invention relates to(a) an antibody, polynucleotide, vector, recombinant host cell, and/orpharmaceutical composition of the present invention and (b) a vaccinefor use as a medicament, for example, for use in a method for thetreatment of cancer, optionally wherein the vaccine comprises a heatshock protein peptide complex (HSPPC) comprising a heat shock proteincomplexed with an antigenic peptide. In one aspect, the presentinvention relates to a pharmaceutical composition, kit or kit-of-partscomprising (a) an antibody, polynucleotide, vector, recombinant hostcell, and/or pharmaceutical composition of the present invention and (b)a vaccine, optionally wherein the vaccine comprises a heat shock proteinpeptide complex (HSPPC) comprising a heat shock protein complexed withan antigenic peptide.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are a set of histograms showing the binding ofanti-LAG-3 Fabs to wild type Jurkat cells or Jurkat cells engineered toexpress human LAG-3, as measured by flow cytometry. The anti-LAG-3 Fabstested in this study include P01A12, P01C09, PO5E01, P13A04, P13A06,P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05,P13E02, P13F01, P13F02, P13F06, P13F09, P13G01, P13G04, P13G05, P13H05,P14A04, P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15CO2,P15E06, P15F06, P15G05, P16D04, and P16H05.

FIGS. 2A and 2B are graphs showing results from assays testing theability of anti-LAG-3 Fabs or a negative control Fab not specific forLAG-3 to block the binding of cross-linked recombinant LAG-3-6His to MHCclass II expressing Raji cells. FIG. 2A is a bar graph showing thepercentage of blocking mediated by the negative control Fab oranti-LAG-3 Fab P13B02, P13C08, P13C10, P13E02, P13F02, P01A12, P13B01,PO5E01, or P01C09. FIG. 2B is a line graph showing the percentage ofLAG-3 binding in the presence of a dose titration of anti-LAG-3 FabP01A12, P13A06, P13B01, P13B02, P13C06, P13C08, P13C10, P13E02, orP14C04, or the negative control Fab.

FIG. 3 is a line graph similar to the one shown in FIG. 2B, in which thepercentage of LAG-3 binding is plotted against a dose titration of fulllength chimeric anti-LAG-3 antibody P13A06, P13B01, P13B02, P13C06,P13C08, or P13E02, or an isotype control antibody.

FIG. 4 is a graph showing the production of IL-2 in human peripheralblood mononuclear cells (PBMCs) upon Staphylococcus Enterotoxin A (SEA)stimulation in the absence of any antibody, or in the presence of anisotype control antibody or the chimeric anti-LAG-3 antibody P13B02.

FIGS. 5A and 5B are sequence alignments of humanized variable regionswith corresponding murine sequences and human germline sequences. FIG.5A is a sequence alignment comparing the humanized heavy chain variableregions H0-H4 (SEQ ID NOs: 56-60, respectively), the murine antibodyP13B02 heavy chain variable region (SEQ ID NO: 15), and the humangermline sequences IGHV1-46*01 (SEQ ID NO: 153) and IGHJ1*01 (SEQ ID NO:200). FIG. 5B is a sequence alignment comparing the humanized lightchain variable regions L0-L4 (SEQ ID NOs: 73-77, respectively), themurine antibody P13B02 light chain variable region (SEQ ID NO: 16), andthe human germline sequences IGKV3-20*01 (SEQ ID NO: 160) and IGKJ1*01(SEQ ID NO: 201). Dots represent residues identical to correspondingresidues in H0 (FIG. 5A) or L0 (FIG. 5B). Dashes represent lack of aminoacid residues compared with H0 (FIG. 5A) or L0 (FIG. 5B).

FIGS. 6A and 6B are graphs showing the binding of anti-LAG-3 antibodiesto human T cells activated by Staphylococcus Enterotoxin A (SEA), asmeasured by flow cytometry. FIG. 6A is a set of histograms testing thechimeric antibody P13B02 (IgG₁), and the humanized antibodies P13B02-06(Ig P13B02-07 (Ig P13B02-16 (Ig P13B02-25 (Ig P13B02-26 (Ig P13B02-27(Ig P13B02-30 (IgG₁ G1m17 N297A), and P13B02-30 (IgG₄). FIG. 6B is agraph showing the binding of the anti-LAG-3 antibody P13B02-16 (IgG₁) oran isotype control antibody to activated primary human CD4+ T cells. Themedian fluorescence intensity (MFI) values are plotted against antibodyconcentrations.

FIGS. 7A and 7B are line graphs similar to the one shown in FIG. 2B, inwhich the percentage of LAG-3 binding is plotted against different dosesof an isotype control antibody, the chimeric antibody P13B02 (IgG₁), thehumanized antibody P13B02-06 (IgG₁), P13B02-07 (Ig P13B02-16 (IgP13B02-26 (IgG₁), or P13B02-27 (IgG₁) (FIG. 7A) or the humanizedantibody P13B02-30 (IgG₁ G1m17 N297A) (FIG. 7B).

FIGS. 8A and 8B are graphs showing the production of IL-2 induced byanti-LAG-3 antibody or isotype control antibody in human peripheralblood mononuclear cells (PBMCs) upon Staphylococcus Enterotoxin A (SEA)stimulation. In FIG. 8A, the anti-LAG-3 antibody tested is P13B02-30(IgG₁). In FIG. 8B, the anti-LAG-3 antibody P13B02-16 (IgG₁) or anisotype control antibody was tested in the presence or absence ofanti-PD-1 antibody pembrolizumab (Pembro) or nivolumab (Nivo),anti-PD-L1 antibody #1, #2, or #3, or anti-CTLA-4 antibody ipilimumab(Ipi).

FIGS. 9A and 9B are graphs showing TNFα production of primary tumorinfiltrating lymphocytes (TILs) induced by anti-LAG-3 antibody P13B02-30(IgG₁ Glm3 N297A) or an isotype control antibody, either alone or incombination with the anti-PD-1 antibody pembrolizumab (Pembro). The TILswere isolated from renal cell carcinoma (FIG. 9A) or colorectal cancer(FIG. 9B) tumors and activated with anti-CD3/CD28 microbeads.

FIGS. 10A and 10B are graphs showing that the anti-LAG-3 antibodyP13B02-30 (IgG₁ Glm3 N297A) enhanced T cell activation in aLAG-3-mediated cell suppression assay. Jurkat-NFAT-luciferase-LAG-3cells were incubated in the presence of sextuplet dose titrations ofeither an anti-LAG-3 antibody (black dots) or an isotype controlantibody (white dots), a fixed concentration of Raji cells, and a fixedconcentration of Staphylococcal Enterotoxin E (SEE) peptide. In a firstexperiment, antibody concentrations between 0.2-50 μg/mL were tested(FIG. 10A). In a second experiment, antibody concentrations between0.1-100 μg/mL were tested (FIG. 10B). RLU=relative light units ofluciferase reporter.

6. DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toLAG-3 (e.g., human LAG-3) and antagonize LAG-3 function, e.g.,LAG-3-mediated immune suppression. Also provided are pharmaceuticalcompositions comprising these antibodies, nucleic acids encoding theseantibodies, expression vectors and host cells for making theseantibodies, and methods of treating a subject using these antibodies.The antibodies disclosed herein are particularly useful for increasing Tcell activation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen), and hence for treating cancer in a subjector treating or preventing an infectious disease in a subject. Allinstances of “isolated antibodies” described herein are additionallycontemplated as antibodies that may be, but need not be, isolated. Allinstances of “isolated polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated. All instances of “antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated.

6.1 Definitions

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., upto 5% to 10% below) the value or range remain within the intendedmeaning of the recited value or range.

As used herein, the term “LAG-3” refers to Lymphocyte activation gene 3(also known as CD223). As used herein, the term “human LAG-3” refers toa human LAG-3 protein encoded by a wild type human LAG-3 gene, e.g.,GenBank™ accession number NM 002286.5. An exemplary immature amino acidsequence of human LAG-3 is provided as SEQ ID NO: 166. Exemplary matureamino acid sequences of human LAG-3 are provided as SEQ ID NO: 167 andSEQ ID NO: 210.

As used herein, the terms “antibody” and “antibodies” include fulllength antibodies, antigen-binding fragments of full length antibodies,and molecules comprising antibody CDRs, VH regions or VL regions.Examples of antibodies include monoclonal antibodies, recombinantlyproduced antibodies, monospecific antibodies, multispecific antibodies(including bispecific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, murine antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer, anantibody heavy chain monomer, an antibody light chain dimer, an antibodyheavy chain dimer, an antibody light chain-antibody heavy chain pair,intrabodies, heteroconjugate antibodies, antibody-drug conjugates,single domain antibodies, monovalent antibodies, single chain antibodiesor single-chain Fvs (scFv), camelized antibodies, affybodies, Fabfragments, F(ab′)₂ fragments, disulfide-linked Fvs (sdFv),anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Idantibodies), and antigen-binding fragments of any of the above. Incertain embodiments, antibodies described herein refer to polyclonalantibody populations. Antibodies can be of any type (e.g., IgG, IgE,IgM, IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG₂a or IgG₂b) of immunoglobulinmolecule. In certain embodiments, antibodies described herein are IgGantibodies, or a class (e.g., human IgG₁ or IgG₄) or subclass thereof.In a specific embodiment, the antibody is a humanized monoclonalantibody. In another specific embodiment, the antibody is a humanmonoclonal antibody.

As used herein, the terms “VH region” and “VL region” refer to singleantibody heavy and light chain variable regions, respectively,comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (ComplementarityDetermining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest (NIH Publication No. 91-3242,Bethesda), which is herein incorporated by reference in its entirety).

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are herein incorporated by reference in theirentireties, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. In certainembodiments, the term “CDR” is a CDR as defined by Kabat et al., J.Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteinof immunological interest. (1991). In certain embodiments, the term“CDR” is a CDR as defined by Chothia et al., J. Mol. Biol. 196:901-917(1987). In certain embodiments, the term “CDR” is a CDR as defined byMacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A.“Protein Sequence and Structure Analysis of Antibody Variable Domains,”in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001).

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunoglobulin chain.The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat or Chothia definition of CDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 125amino acids in the mature heavy chain and about 90 to 115 amino acids inthe mature light chain, which differ extensively in sequence amongantibodies and are used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen. In certain embodiments, thevariable region is a human variable region. In certain embodiments, thevariable region comprises rodent or murine CDRs and human frameworkregions (FRs). In particular embodiments, the variable region is aprimate (e.g., non-human primate) variable region. In certainembodiments, the variable region comprises rodent or murine CDRs andprimate (e.g., non-human primate) framework regions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light and/orheavy chain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with an Fc receptor (e.g., Fc gamma receptor). The constantregion of an immunoglobulin molecule generally has a more conservedamino acid sequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al., Sequences of Proteins of ImmunologicalInterest, U.S. Dept. Health and Human Services, 5th edition, 1991, eachof which is herein incorporated by reference in its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation rateconstant of, e.g., an antibody to an antigen. The k_(on), and k_(off)can be determined by techniques known to one of ordinary skill in theart, such as BIAcore® or KinExA. As used herein, a “lower affinity”refers to a larger K_(D).

As used herein, the terms “specifically binds,” “specificallyrecognizes,” “immunospecifically binds,” and “immunospecificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, Id.), or other assays known in the art. In aspecific embodiment, molecules that specifically bind to an antigen bindto the antigen with a K_(A) that is at least 2 logs (i.e., factors of10), 2.5 logs, 3 logs, 4 logs or greater than the K_(A) when themolecules bind non-specifically to another antigen.

In another specific embodiment, molecules that specifically bind to anantigen do not cross react with other proteins under similar bindingconditions. In another specific embodiment, molecules that specificallybind to LAG-3 do not cross react with other non-LAG-3 proteins. In aspecific embodiment, provided herein is an antibody that binds to LAG-3(e.g., human LAG-3) with higher affinity than to another unrelatedantigen. In certain embodiments, provided herein is an antibody thatbinds to LAG-3 (e.g., human LAG-3) with a 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity thanto another, unrelated antigen as measured by, e.g., a radioimmunoassay,surface plasmon resonance, or kinetic exclusion assay. In a specificembodiment, the extent of binding of an anti-LAG-3 antibody describedherein to an unrelated, non-LAG-3 protein is less than 10%, 15%, or 20%of the binding of the antibody to LAG-3 protein as measured by, e.g., aradioimmunoassay.

As used herein, the term “afucosylation” or “afucosylated” in thecontext of an Fc refers to a substantial lack of a fucose covalentlyattached, directly or indirectly, to residue 297 of the human IgG₁ Fcregion, numbered according to the EU numbering system, or thecorresponding residue in non-IgG₁ or non-human IgG₁ immunoglobulins.Thus, in a composition comprising a plurality of afucosylatedantibodies, at least 70% of the antibodies will not be fucosylated,directly or indirectly (e.g., via intervening sugars) at residue 297 ofthe Fc region of the antibodies, and in some embodiments at least 80%,85%, 90%, 95%, or 99% will not be fucosylated, directly or indirectly,at residue 297 of the Fc region.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays (e.g., constraining peptides using CLIPS (Chemical Linkage ofPeptides onto Scaffolds) to map discontinuous or conformationalepitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of whichare herein incorporated by reference in their entireties).Antibody:antigen crystals may be studied using well known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H Wet al.; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) ActaCrystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) MethEnzymol 276A: 361-423, ed Carter C W; Roversi P et al., (2000) ActaCrystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), all of which areherein incorporated by reference in their entireties. Mutagenesismapping studies may be accomplished using any method known to one ofskill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham B C & Wells J A (1989) Science 244: 1081-1085,each of which is herein incorporated by reference in its entirety, for adescription of mutagenesis techniques, including alanine scanningmutagenesis techniques. CLIPS (Chemical Linkage of Peptides ontoScaffolds) is a technology to present one or more peptides in astructurally constrained configuration to behave as functional mimics ofcomplex protein domains. See, e.g., U.S. Publication Nos. US2008/0139407 A1 and US 2007/099240 A1, and U.S. Pat. No. 7,972,993, eachof which is herein incorporated by reference in its entirety. In aspecific embodiment, the epitope of an antibody is determined usingalanine scanning mutagenesis studies. In a specific embodiment, theepitope of an antibody is determined using hydrogen/deuterium exchangecoupled with mass spectrometry. In a specific embodiment, the epitope ofan antibody is determined using CLIPS Epitope Mapping Technology fromPepscan Therapeutics.

As used herein, the term “an epitope located within a region of humanLAG-3” consisting of a particular amino acid sequence or a set of aminoacid residues refers to an epitope comprising one or more of the aminoacid residues of the specified region, wherein the specified regionincludes the first specified amino acid residue and the last specifiedamino acid residue of the region of human LAG-3. In certain embodiments,the epitope comprises each one of the amino acid residues located withinthe specified region. In certain embodiments, one or more additionalamino acid residues of human LAG-3 outside the specified region bind toan antibody together with an epitope located within the specifiedregion.

As used herein, the terms “T cell receptor” and “TCR” are usedinterchangeably and refer to full length heterodimeric αβ or γδ TCRs,antigen-binding fragments of full length TCRs, and molecules comprisingTCR CDRs or variable regions. Examples of TCRs include, but are notlimited to, full length TCRs, antigen-binding fragments of full lengthTCRs, soluble TCRs lacking transmembrane and cytoplasmic regions,single-chain TCRs containing variable regions of TCRs attached by aflexible linker, TCR chains linked by an engineered disulfide bond,monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCRfusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantlyproduced TCRs, and synthetic TCRs. The term encompasses wild-type TCRsand genetically engineered TCRs (e.g., a chimeric TCR comprising achimeric TCR chain which includes a first portion from a TCR of a firstspecies and a second portion from a TCR of a second species).

As used herein, the terms “major histocompatibility complex” and “MHC”are used interchangeably and refer to an MHC class I molecule and/or anMHC class II molecule.

As used herein, the term “peptide-MHC complex” refers to an MHC molecule(MHC class I or MHC class II) with a peptide bound in the art-recognizedpeptide binding pocket of the MHC.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration of an antibody to a subject having adisease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the term “subject” includes any human or non-humananimal. In one embodiment, the subject is a human or non-human mammal.In one embodiment, the subject is a human.

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877, eachof which is herein incorporated by reference in its entirety. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403, which is hereinincorporated by reference in its entirety. BLAST nucleotide searches canbe performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules described herein. BLAST protein searches canbe performed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule described herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul S F etal., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated byreference in its entirety. Alternatively, PSI BLAST can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blastprograms, the default parameters of the respective programs (e.g., ofXBLAST and NBLAST) can be used (see, e.g., National Center forBiotechnology Information (NCBI) on the worldwide web,ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is hereinincorporated by reference in its entirety. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

6.2 Anti-LAG-3 Antibodies

In one aspect the instant disclosure provides antibodies thatspecifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3function. The amino acid sequences of exemplary antibodies are set forthin Tables 1-7 herein.

The skilled worker will appreciate that the N-terminal E or Q amino acidresidue can, under certain conditions, spontaneously convert topyroglutamate by post-translational cyclization of the free amino groupto form a lactam. Accordingly, in certain embodiments, the instantdisclosure provides antibodies comprising an antibody heavy chainvariable region or light chain variable region disclosed herein (e.g.SEQ ID NOs: 56-72 and 73-77, respectively) or full length heavy chain orlight chain disclosed herein (e.g. SEQ ID NOs: 168-186 and 187-191,respectively), wherein the N-terminal E or Q amino acid residue has beenconverted to pyroglutamate (e.g., as a result of post-translationalcyclization of the free amino group of the N-terminal E or Q residue).

TABLE 1 Amino acid sequences of exemplary anti-LAG-3 antibodies. SEQID NO: Description Amino acid sequence 1 P01C09 VHQVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDPKLQGKATITADTSSNTVYLQLSSLTSEDTAVFYCVIYSYRYDVGGFDYW GQGTTLTVS 2 P01C09 VL, P13F01 VL,EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWY P13G05 VL, P05E03 VLQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK3 P05E01 VH DVQLVESGAELVKPGASVKLSCTASGFTIKDTYIHWVKQRPEQGLEWIGEIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 4 P05E01 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISNMEAEDAATYYCQQWNSYPLTFGAGTKLELK5 P01A12 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLDWIGRIDPANGNTKFDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSTYYYRYDVGGFDYW GQGTTLTVS 6 P01A12 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK7 P13B01 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPERGLEWIGRIDPANGNTKFDPKFQGTATITADTSSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 8 P13B01 VL, P14C04 VL,ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWY P14B07 VLQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK9 P13C10 VH, P15C02 VH, QVQLQQSGAELVKPGASVELSCTASGFNIRDTYMYWVP16D04 VH, P13G05 VH, KQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTP13F06 VH, P14F01 VH, SSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW P16H01 VHGQGTTLTVS 10 P13C10 VL, P13E02 VL, ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFP13F02 VL, P13B03 VL, QQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLP13H05 VL, P13G04 VL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 11 P13C08 VHQVQLKQSGAELVKPGASVKLSCTASGFNIKDNYIHWVKQRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYW GQGTTLTVS 12 P13C08 VLDVVMTQTPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK13 P13E02 VH QVQLQQPGAELVKPGASVKLSCTVSGFNIKDTYIHWVKQRPEQGLEWIGEIDPANGNSKYAPRFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 14 P13F02 VHQVQLQQPGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLDWVGEIDPANGHTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 15 P13B02 VHQVQMKQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGEIDPANDNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 16 P13B02 VLEILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK17 P13A06 VH QVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTVYLQLSSLTSEDTAVFYCVIYSYRYDVGGFDYW GQGTTLTVS 18 P13A06 VLENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPHTFGGGTKLEIK19 P14C04 VH QVQLQQPGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLDWIGRIDPANGNTHFDPKFQGKATITADTSSNTAYLQLSSLISEDTAVYYCSTYFYRYDVGGFDYW GQGTTLTVS 20 P14A04 VH, P13F01 VH,QVQLQQPGAELVKPGASVELSCTASGFNIRDTYMYWV P15E06 VHKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLISEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 21 P14A04 VL, P14G01 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK22 P15F06 VH QVQLKQSGAELVKPGASVELSCTASGFNIRDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLISEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 23 P15F06 VLENVLTQSPALMAASPGEKVTITCSVSSSISSSTLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPWTFGGGTKLEIK24 P13B03 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGEIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 25 P15C02 VLDVVMTQTPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK26 P16D04 VL, P16H01 VL ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPLTFGAGTKLELK27 P13A04 VH QVQLQQPGAELVKPGASVELSCTASGFNIRDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSADTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 28 P13A04 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISNMEAEDAATYYCQQWSSYPFTFGSGTKLEIK29 P16H05 VH QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYIHWVKQRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYW GQGTTLTVS 30 P16H05 VLEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPHTFGGGTKLEIK31 P13F09 VH QVQLQQSGAELVKPGASVKLSCTASGFNIKDNYIHWVKQRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSSNTAYLQLSSLTSEGTAVYYCASYFYRYDVGGFDYW GQGTTLTVS 32 P13F09 VL, P14F06 VLEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK33 P13G01 VH, P15G05 VH EVQLQQSGAELVKPGASVELSCTASGFNIRDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 34 P13G01 VLQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK35 P13H05 VH QVQLQQPGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGEIDPANDNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 36 P13D04 VHQVQLQQSGAELVKPGASVKLSCTASGFNIKDNYMDWVKQRPEQGLEWIGKIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 37 P13D04 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTPPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK38 P14G01 VH, P05E03 VH QVQLKESGAELVKPGASVELSCTASGFNIRDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 39 P14G03 VHQVQMKQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPGQGLEWIGEIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 40 P14G03 VLDIVLTQSPALMAASPGEKVTITCSVSSSISSSNLYWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK41 P13F06 VL EILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK42 P13B11 VH QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYMDWVKQRPEQGLEWIGKIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYW GQGTTLTVS 43 P13B11 VLQIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK44 P14F01 VL ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSTYPFTFGSGTKLEIK45 P14F06 VH QVQMKQSGAELVKPGASVKLSCTASGFNIKDNYIHWVKQRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYW GQGTTLTVS 46 P13D05 VHQVQLQQPGAELVKPGASVELSCTASGFNIRDTYMYWVKQRPEQGLGWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 47 P13D05 VLEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFRQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK48 P13G04 VH EVKLMESGAELVKPGASAELSCTASGFNIRDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYW GQGTTLTVS 49 P15E06 VLENVLTQSPALMAASPGEKVTITCSVSSGISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSRYPWTFGGGTKLEIK50 P15G05 VL EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPLTFGAGTKLELK51 P15B06 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLDWIGRIDPANGNTHFDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSTYFYRYDVGGFDYW GQGTTLTVS 52 P15B06 VLQILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL TISSMEAEDAATYYCQQWSSYPLTFGAGTKLELK53 P14B07 VH QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYIHWVKQRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSSNTAYLQLSSLTSEDTTVYYCASYFYRYDVGGFDYW GQGTTLTVS 54 P13C06 VHQVQMKQSGAELVKPGASVELSCTASGFNITDTYMYWVKQRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSSNTAYLQLSSLTSEDTAVYYCTTYFDKYDVGGCDYW GQGTTLTVS 55 P13C06 VLEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLYWFQHKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSL IISSMDAENAATYYCQQWRSYPFTFGSGTKLEIK56 H0 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTMTRDTSISTVYMELSSLRSEDTAVYYCARYYYRYDVGGFDYW GQGTLVTVSS 57 H1QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYW GQGTLVTVSS 58 H2QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYW GQGTLVTVSS 59 H3QVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYW GQGTLVTVSS 60 H4QVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYRYDVGGFDYW GQGTLVTVSS 61 H4_R98KQVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 62 H4_D100EQVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYRYEVGGFDYW GQGTLVTVSS 63 H1_R98KQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 64 H1_R98K_K23TQVQLVQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 65 H1_R98K_L4MQVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 220 H1_R98K_L4MXVQMVQSGAEVKKPGASVKVSCKASGFNIKD TYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS,wherein X = glutamine (Q) or pyroglutamate (pE) 66 H1_R98K_L4M_K23TQVQMVQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 67 H1_R98K_L4M_V5KQVQMKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 68 H1_R98K_L4M_V5K_QVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV K23TRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 69 H1_R98K_V5KQVQLKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 70 H1_R98K_V5K_K23TQVQLKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 71 H2_R98KQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 72 H3_R98KQVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYW GQGTLVTVSS 73 L0EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK221 L0 XIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK,wherein X = glutamate (E) or pyroglutamate (pE) 74 L1EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTL TISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK75 L2 EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTL TISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK76 L3 EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQQKPGQAPRLWIYGTSNLASGVPDRFSGSGSGTSYTL TISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK77 L4 EILLTQSPGTLSLSPGERATLTCSVSSSISSSNLHWFQQKPGQSPRLWIYGTSNLASGVPDRFSGSGSGTSYTL TISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK78 CDRH1 DTYMY 79 CDRH1 DTYIH 80 CDRH1 DTYIY 81 CDRH1 DNYIH 82 CDRH1DNYMD 83 CDRH2 RIDPANGNTKYDPKLQG 84 CDRH2 EIDPANGNTKYDPKFQG 85 CDRH2RIDPANGNTKFDPKFQG 86 CDRH2 RIDPANGNTKFDPKFQD 87 CDRH2 SIDPANGNTKYDPKFQG88 CDRH2 EIDPANGNSKYAPRFQG 89 CDRH2 EIDPANGHTKYDPKFQG 90 CDRH2EIDPANDNTKYDPKFQG 91 CDRH2 RIDPANGNTKYDPKFQG 92 CDRH2 RIDPANGNTHFDPKFQG93 CDRH2 KIDPANGNTKYDPKFQG 94 CDRH3 YSYRYDVGGFDY 95 CDRH3 YYYRYDVGGFDY96 CDRH3 YFYRYDVGGFDY 97 CDRH3 YFDKYDVGGCDY 98 CDRH3 YYYKYDVGGFDY 99CDRH3 YYYRYEVGGFDY 100 CDRL1 SVSSSISSSNLH 101 CDRL1 SVSSSISSSTLH 102CDRL1 SVSSSISSSNLY 103 CDRL1 SVSSGISSSNLH 104 CDRL2 GTSNLAS 105 CDRL3QQWSSYPFT 106 CDRL3 QQWNSYPLT 107 CDRL3 QQWSSYPHT 108 CDRL3 QQWSSYPWT109 CDRL3 QQWSSYPLT 110 CDRL3 QQWSTYPFT 111 CDRL3 QQWSRYPWT 112 CDRL3QQWRSYPFT 113 VH FR1 QVQLVQSGAEVKKPGASVKVSCKASGFNIK 114 VH FR1QVQMKQSGAEVKKPGASVKVSCTASGFNIK 115 VH FR1 QVQMKQSGAEVVKPGASVKVSCTASGFNIK116 VH FR1 QVQLVQSGAEVKKPGASVKVSCTASGFNIK 117 VH FR1QVQMVQSGAEVKKPGASVKVSCKASGFNIK 118 VH FR1 QVQMVQSGAEVKKPGASVKVSCTASGFNIK119 VH FR1 QVQMKQSGAEVKKPGASVKVSCKASGFNIK 120 VH FR1QVQLKQSGAEVKKPGASVKVSCKASGFNIK 121 VH FR1 QVQLKQSGAEVKKPGASVKVSCTASGFNIK122 VH FR2 WVRQAPGQGLEWMG 123 VH FR2 WVRQAPGQGLEWIG 124 VH FR3RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 125 VH FR3RVTITADTSTSTVYMELSSLRSEDTAVYYCAT 126 VH FR3RVTITADTSSNTVYMELSSLRSEDTAVYYCAT 127 VH FR3RVTITADTSSNTVYLQLSSLRSEDTAVYYCAT 128 VH FR4 WGQGTLVTVSS 129 VL FR1EIVLTQSPGTLSLSPGERATLSC 130 VL FR1 EILLTQSPGTLSLSPGERATLSC 131 VL FR1EILLTQSPGTLSLSPGERATLTC 132 VL FR2 WYQQKPGQAPRLLIY 133 VL FR2WFQQKPGQAPRLLIY 134 VL FR2 WFQQKPGQAPRLWIY 135 VL FR2 WFQQKPGQSPRLWIY136 VL FR3 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 137 VL FR3GIPDRFSGSGSGTSYTLTISRLEPEDFAVYYC 138 VL FR3GVPDRFSGSGSGTSYTLTISRLEPEDFAVYYC 139 VL FR4 FGQGTKVEIK 140CDRH1 consensus DX₁YX₂X₃, wherein: X₁ is T or N; X₂ is I or M; andX₃ is H, Y or D 141 CDRH1 consensus DX₁YX₂X₃, wherein: X₁ is T or N;X₂ is I or M; and X₃ is H or Y 142 CDRH2 consensusX₁IDPANX₂X₃X₄X₅X₆X₇PX₈X₉QX₁₀, wherein: X₁ is E, R, S, or K;X₂ is D or G; X₃ is N or H; X₄ is T or S; X₅ is K or H; X₆ is Y or F;X₇ is D or A; X₈ is K or R; X₉ is F or L; and X₁₀ is G or D 143CDRH2 consensus X₁IDPANX₂X₃X₄KX₅X₆PX₇FQX₈, wherein: X₁ is E, R, or S;X₂ is D or G; X₃ is N or H; X₄ is T or S; X₅ is Y or F; X₆ is D or A;X₇ is K or R; and X₈ is G or D 144 CDRH3 consensusYX₁X₂X₃YX₄VGGX₅DY, wherein: X₁ is Y, F, or S; X₂ is Y or D;X₃ is K or R; X₄ is D or E; and X₅ is F or C 145 CDRH3 consensusYX₁X₂X₃YDVGGX₄DY, wherein: X₁ is Y, F, or S; X₂ is Y or D;X₃ is K or R; and X₄ is F or C 146 CDRH3 consensusYYYX₁YX₂VGGFDY, wherein: X₁ is K or R; and X₂ is D or E 147CDRL1 consensus SVSSX₁ISSSX₂LX₃, wherein: X₁ is S or G;X₂ is N or T; and X₃ is H or Y 148 CDRL1 consensusSVSSSISSSNLX₁, wherein: X₁ is H or Y 149 CDRL3 consensusQQWX₁X₂YPX₃T, wherein: X₁ is S, N, or R; X₂ is S, T or R; andX₃ is F, L, H, or W 150 CDRL3 consensus QQWX₁SYPX₂T, wherein:X₁ is S, N, or R; and X₂ is F, L, or H 151 Humanized VH consensusQVQX₁X₂QSGAEVX₃KPGASVKVSCKASGFNIKDTYTHWVRQAPGQGLEWX₅GEIDPANDNTKYDPKFQGRVTX₆TX₇DTSX₈X₉TVYX₁₀X₁₁LSSLRSEDTAVYYCAX₁₂YYYX₁₃YX₁₄VGGFDYWGQGTLVTVSS, wherein: X₁ is M or L; X₂ is V or K;X₃ is K or V; X₄ is K or T; X₅ is M or I; X₆ is I or M; X₇ is A or R;X₈ is T or S; X₉ is S or N; X₁₀ is M or L; X₁₁ is E or Q; X₁₂ is T or R;X₁₃ is K or R; and X₁₄ is D or E 218 Humanized VH consensusQVQX₁X₂QSGAEVX₃KPGASVKVSCX₄ASGFNIKDTYTHWVRQAPGQGLEWX₅GEIDPANDNTKYDPKFQGRVTX₆TX₇DTSX₈X₉TVYX₁₀X₁₁LSSLRSEDTAVYYCAX₁₂YYYX₁₃YX₁₄VGGFDYWGQGTLVTVSS, wherein: X₁ is M or L; X₂ is V or K;X₃ is K or V; X₄ is K or T; X₅ is M or I; X₆ is I or M; X₇ is A or R;X₈ is T or S; X₉ is S or N; X₁₀ is M or L; X₁₁ is E or Q; X₁₂ is T or R;X₁₃ is K or R; and X₁₄ is D or E 222 Humanized VH consensusX₁VQX₂X₃QSGAEVX₄KPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWX₅GEIDPANDNTKYDPKFQGRVTX₆TX₇DTSX₈X₉TVYX₁₀X₁₁LSSLRSEDTAVYYCAX₁₂YYYX₁₃YX₁₄VGGFDYWGQGTLVTVSS, wherein: X₁ is Q or pE (Pyroglutamate)X₂ is M or L; X₃ is V or K; X₄ is K or V; X₅ is M or I; X₆ is I or M;X₇ is A or R; X₈ is T or S; X₉ is S or N; X₁₀ is M or L; X₁₁ is E or Q;X₁₂ is T or R; X₁₃ is K or R; and X₁₄ is D or E 223Humanized VH consensus X₁VQX₂X₃QSGAEVX₄KPGASVKVSCX₅ASGFNIKDTYIHWVRQAPGQGLEWX₆GEIDPANDNTKYDPKFQGRVTX₇TX₈DISX₉X₁₀TVYX₁₁X₁₂LSSLRSEDTAVYYCAX₁₃YYYX₁₄YX₁₅VGGFDYWGQGTLVTVSS, wherein: X₁ is Q or pE (Pyroglutamate)X₂ is M or L; X₃ is V or K; X₄ is K or V; X₅ is K or T; X₆ is M or I;X₇ is I or M; X₈ is A or R; X₉ is T or S; X₁₀ is S or N; X₁₁ is M or L;X₁₂ is E or Q; X₁₃ is T or R; X₁₄ is K or R; and X₁₅ is D or E 152Humanized VLconsensus EIX₁LTQSPGILSLSPGERATLX₂CSVSSSISSSNLHWX₃QQKPGQX₄PRLX₅IYGTSNLASGX₆PDRFSGSGSGTX₇X₈TLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEI K, wherein: X₁ is V or L;X₂ is S or T; X₃ is Y or F; X₄ is A or S; X₅ is L or W; X₆ is I or V;X₇ is D or S; and X₈ is F or Y 224 Humanized VLconsensusX₁IX₂LTQSPGILSLSPGERATLX₃CSVSSSISSSNLHWX₄QQKPGQX₅PRLX₆IYGTSNLASGX₇PDRFSGSGSGTX₈X₉TLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVE IK, wherein:X₁ is E or pE(Pyroglutamate) X₂ is V or L; X₃ is S or T; X₄ is Y or F;X₅ is A or S; X₆ is L or W; X₇ is I or V; X₈ is D or S; and X₉ is F or Y168 H1_R98K_L4M full QVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVlength IgG₁ heavy chain RQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSTSTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 225 H1_R98K_L4M_fullXVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₁ heavy chainRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG,wherein X = glutamine (Q) or pyroglutamate (pE) 169 H1_R98K_L4M_fullQVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₁ N297A heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 226 H1_R98K_L4M_fullXVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₁ N297A heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG,wherein X = glutamine (Q) or pyroglutamate (pE) 170 H1_R98K_L4M_fullQVQMVQSGAEVKKPGASVKVSCKASGFNIKD TYIHWV length IgG₄ S228P heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG 227 H1_R98K_L4M_fullXVQMVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₄ S228P heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSTSTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG,wherein X = glutamine (Q) or pyroglutamate (pE) 171 HO full length IgG₁QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV N297A heavy chainRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTMTRDTSISTVYMELSSLRSEDTAVYYCARYYYRYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 172 H1 full length IgG₁QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV N297A heavy chainRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 173 H2 full length IgG₁QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV N297A heavy chainRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 174 H3 full length IgG₁QVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV N297A heavy chainRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 175 H4 full length IgG₁QVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWV N297A heavy chainRQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYRYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 176H4_R98K full length IgG₁ QVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWVN297A heavy chain RQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 177 H4_D100E full lengthQVQMKQSGAEVVKPGASVKVSCTASGFNIKDTYIHWV IgG₁ N297A heavy chainRQAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSSNTVYLQLSSLRSEDTAVYYCATYYYRYEVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 178H1_R98K full length IgG₁ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVN297A heavy chain RQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 179 H1_R98K_K23T fullQVQLVQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV length IgG₁ N297A heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 180 H1_R98K_L4M_K23TQVQMVQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV full length IgG₁ N297ARQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT heavy chainSTSTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 181 H1_R98K_L4M_V5K fullQVQMKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₁ N297A heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 182 H1_R98K_L4M_V5K_KQVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV 23T full length IgG₁RQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT N297A heavy chainSTSTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 183 H1_R98K_V5K_fullQVQLKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWV length IgG₁ N297A heavyRQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT chainSISTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 184 H1_R98K_V5K_K23TQVQLKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWV full length IgG₁ N297ARQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT heavy chainSTSTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 185H2_R98K full length IgG₁ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVN297A heavy chain RQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 186H3_R98K full length IgG₁ QVQMKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVN297A heavy chain RQAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTSSNTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 187L0 full length light chain EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTLIISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 228L0 full length light chain XIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC,wherein X = glutamate (E) or pyroglutamate (pE) 188L1 full length light chain EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 189L2 full length light chain EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQQKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 190L3 full length light chain EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQQKPGQAPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 191L4 full length light chain EILLTQSPGTLSLSPGERATLTCSVSSSISSSNLHWFQQKPGQSPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 192Human IgG₁ Glm3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVallotype (without C- TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSterminal lysine) SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG193 Human IgG₁ Glm3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV allotypeTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK208 Human IgG₁ Glm17 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVallotype (without C- TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSterminal lysine) SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG209 Human IgG₁ Glm17 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV allotypeTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK194 IgG₁ N297A (without C- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVterminal lysine) TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG195 IgG₁ N297A ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK196 IgG₄ S228P (without C- ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVterminal lysine) TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLG 197IgG₄ S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK198 Human kappa light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAconstant region IGKC*01 KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLKm3 allotype SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 219Human kappa light chain GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAconstant region IGKC*01 KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLKm3 allotype SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 2 Heavy chain CDR amino acid sequences of exemplary anti-LAG-3 antibodies.* CDRH1 (SEQ ID CDRH2 CDRH3 VII NO:) (SEQ ID NO:)(SEQ ID NO:) P01C09 VH DTYMY (78) RIDPANGNTKYDPKLQG (83)YSYRYDVGGFDY (94) P05E01 VH DTYIH (79) EIDPANGNTKYDPKFQG (84)YYYRYDVGGFDY (95) P01Al2 VH DTYMY (78) RIDPANGNTKFDPKFQG (85)YYYRYDVGGFDY (95) P13B01 VH DTYIY (80) RIDPANGNTKFDPKFQG (85)YFYRYDVGGFDY (96) P13C10 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13C08 VH DNYIH (81) SIDPANGNTKYDPKFQG (87)YFYRYDVGGFDY (96) P13E02 VH DTYIH (79) EIDPANGNSKYAPRFQG (88)YYYRYDVGGFDY (95) P13F02 VH DTYIH (79) EIDPANGHTKYDPKFQG (89)YYYRYDVGGFDY (95) P13B02 VH DTYIH (79) EIDPANDNTKYDPKFQG (90)YYYRYDVGGFDY (95) P13A06 VH DTYMY (78) RIDPANGNTKYDPKFQG (91)YSYRYDVGGFDY (94) P14C04 VH DTYMY (78) RIDPANGNTHFDPKFQG (92)YFYRYDVGGFDY (96) P14A04 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P15F06 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13B03 VH DTYIH (79) EIDPANGNTKYDPKFQG (84)YYYRYDVGGFDY (95) P15C02 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P16D04 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13F01 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13A04 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P16H05 VH DNYIH (81) SIDPANGNTKYDPKFQG (87)YFYRYDVGGFDY (96) P13F09 VH DNYIH (81) SIDPANGNTKYDPKFQG (87)YFYRYDVGGFDY (96) P13G01 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13H05 VH DTYIH (79) EIDPANDNTKYDPKFQG (90)YYYRYDVGGFDY (95) P13D04 VH DNYMD (82) KIDPANGNTKYDPKFQG (93)YYYRYDVGGFDY (95) P14G01 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P14G03 VH DTYIH (79) EIDPANGNTKYDPKFQG (84)YYYRYDVGGFDY (95) P13G05 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13F06 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13B11 VH DNYMD (82) KIDPANGNTKYDPKFQG (93)YYYRYDVGGFDY (95) P14F01 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P14F06 VH DNYIH (81) SIDPANGNTKYDPKFQG (87)YFYRYDVGGFDY (96) P13D05 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13G04 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P15E06 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P15G05 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P15B06 VH DTYMY (78) RIDPANGNTHFDPKFQG (92)YFYRYDVGGFDY (96) P14B07 VH DNYIH (81) SIDPANGNTKYDPKFQG (87)YFYRYDVGGFDY (96) P05E03 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFYRYDVGGFDY (96) P13C06 VH DTYMY (78) RIDPANGNTKFDPKFQD (86)YFDKYDVGGCDY (97) H0 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95)H1 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H2 DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H3 DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H4 DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H4_R98K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H4_D100E DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYRYEVGGFDY (99) H1_R98K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_K23T DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_L4M DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_L4M_K23T DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_L4M_V5K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_L4M_V5K_K23T DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_V5K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H1_R98K_V5K_K23T DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H2_R98K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) H3_R98K DTYIH (79)EIDPANDNTKYDPKFQG (90) YYYKYDVGGFDY (98) *Defined according to the Kabatnumbering system.

TABLE 3 Light chain CDR amino acid sequencesof exemplary anti-LAG-3 antibodies.* CDRL2 CDRL3 CDRL1 (SEQ (SEQ VL(SEQ ID NO:) ID NO:) ID NO:) P01C09 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT(100) (104) (105) P05E01 VL SVSSSISSSNLH GTSNLAS QQWNSYPLT (100) (104)(106) P01A12 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105)P13B01 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P13C10 VLSVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P13C08 VL SVSSSISSSNLHGTSNLAS QQWSSYPFT (100) (104) (105) P13E02 VL SVSSSISSSNLH GTSNLASQQWSSYPFT (100) (104) (105) P13F02 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT(100) (104) (105) P13B02 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104)(105) P13A06 VL SVSSSISSSNLH GTSNLAS QQWSSYPHT (100) (104) (107)P14C04 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P14A04 VLSVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P15F06 VL SVSSSISSSTLHGTSNLAS QQWSSYPWT (101) (104) (108) P13B03 VL SVSSSISSSNLH GTSNLASQQWSSYPFT (100) (104) (105) P15C02 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT(100) (104) (105) P16D04 VL SVSSSISSSNLH GTSNLAS QQWSSYPLT (100) (104)(109) P13F01 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) P13A04 VLSVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P16H05 VL SVSSSISSSNLHGTSNLAS QQWSSYPHT (100) (104) (107) P13F09 VL SVSSSISSSNLH GTSNLASQQWSSYPFT (100) (104) (105) P13G01 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT(100) (104) (105) P13H05 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104)(105) P13D04 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105)P14G01 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P14G03 VLSVSSSISSSNLY GTSNLAS QQWSSYPFT (102) (104) (105) P13G05 VL SVSSSISSSNLHGTSNLAS QQWSSYPFT (100) (104) (105) P13F06 VL SVSSSISSSNLH GTSNLASQQWSSYPFT (100) (104) (105) P13B11 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT(100) (104) (105) P14F01 VL SVSSSISSSNLH GTSNLAS QQWSTYPFT (100) (104)(110) P14F06 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105)P13D05 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P13G04 VLSVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) P15E06 VL SVSSGISSSNLHGTSNLAS QQWSRYPWT (103) (104) (111) P15G05 VL SVSSSISSSNLH GTSNLASQQWSSYPLT (100) (104) (109) P15B06 VL SVSSSISSSNLH GTSNLAS QQWSSYPLT(100) (104) (109) P14B07 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104)(105) P05E03 VL SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105)P13C06 VL SVSSSISSSNLY GTSNLAS QQWRSYPFT (102) (104) (112) L0SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) L1 SVSSSISSSNLH GTSNLASQQWSSYPFT (100) (104) (105) L2 SVSSSISSSNLH GTSNLAS QQWSSYPFT (100)(104) (105) L3 SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) L4SVSSSISSSNLH GTSNLAS QQWSSYPFT (100) (104) (105) *Defined according tothe Kabat numbering system.

TABLE 4 Heavy chain framework (FR) amino acid sequences ofexemplary anti-LAG-3 antibodies.*  VH FR2 VH FR4  VH VH FRI (SEQ IDVH FR3 (SEQ ID  (SEQ ID NO:) NO:) (SEQ ID NO:)  NO:)  H0QVQLVQSGAEVKKPGASV WVRQAPGQGL RVTMTRDTSTSTVYMELSSL WGQGTLVTVKVSCKASGFNIK (113) EWMG (122) RSEDTAVYYCAR (124) SS (128) H1QVQLVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSL WGQGTLVTVKVSCKASGFNIK (113) EWMG (122) RSEDTAVYYCAT (125) SS (128) H2QVQLVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSSNTVYMELSSL) WGQGTLVTVKVSCKASGFNIK (113) EWMG (122) RSEDTAVYYCAT (126 SS (128) H3QVQMKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSSNTVYMELSSL WGQGTLVTVKVSCTASGFNIK (114) EWMG (122) RSEDTAVYYCAT (126) SS (128) H4QVQMKQSGAEWKPGASV WVRQAPGQGL RVTITADTSSNTVYLQLSSL WGQGTLVTVKVSCTASGFNIK (115) EWIG (123) RSEDTAVYYCAT (127) SS (128) H4_R98KQVQMKQSGAEWKPGASV WVRQAPGQGL RVTITADTSSNTVYLQLSSL WGQGTLVTVKVSCTASGFNIK (115) EWIG (123) RSEDTAVYYCAT (127) SS (128) H4_D100EQVQMKQSGAEWKPGASV WVRQAPGQGL RVTITADTSSNTVYLQLSSL WGQGTLVTVKVSCTASGFNIK (115) EWIG (123) RSEDTAVYYCAT (127) SS (128) H1_R98KQVQLVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSL WGQGTLVTVKVSCKASGFNIK (113) EWMG (122) RSEDTAVYYCAT (125) SS (128) H1_R98K_K23TQVQLVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSL WGQGTLVTVKVSCTASGFNIK (116) EWMG (122) RSEDTAVYYCAT (125) SS (128) H1_R98K_L4MQVQMVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSL WGQGTLVTVKVSCKASGFNIK (117) EWMG (122) RSEDTAVYYCAT (125) SS (128)H1_R98K_L4M_K23T QVQMVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSLWGQGTLVTV KVSCTASGFNIK (118) EWMG (122) RSEDTAVYYCAT (125) SS (128)H1_R98K_L4M_V5K QVQMKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSLWGQGTLVTV KVSCKASGFNIK (119) EWMG (122) RSEDTAVYYCAT (125) SS (128)H1_R98K_L4M_V5K_K23T QVQMKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSLWGQGTLVTV KVSCTASGFNIK (114) EWMG (122) RSEDTAVYYCAT (125) SS (128)H1_R98K_V5K QVQLKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSL WGQGTLVTVKVSCKASGFNIK (120) EWMG (122) RSEDTAVYYCAT (125) SS (128)H1_R98K_V5K_K23T QVQLKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSTSTVYMELSSLWGQGTLVTV KVSCTASGFNIK (121) EWMG (122) RSEDTAVYYCAT (125) SS (128)H2_R98K QVQLVQSGAEVKKPGASV WVRQAPGQGL RVTITADTSSNTVYMELSSL WGQGTLVTVKVSCKASGFNIK (113) EWMG (122) RSEDTAVYYCAT (126) SS (128) H3_R98KQVQMKQSGAEVKKPGASV WVRQAPGQGL RVTITADTSSNTVYMELSSL WGQGTLVTVKVSCTASGFNIK (114) EWMG (122) RSEDTAVYYCAT (126) SS (128) *The heavychain framework regions described in Table 4 are determined based uponthe boundaries of the Kabat numbering system for CDRs. In other words,the VH CDRs are determined by Kabat and the framework regions are theamino acid residues surrounding the CDRs in the variable region in theformat FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

TABLE 5 Light chain framework (FR) amino acid sequences ofexemplary anti-LAG-3 antibodies.*  VL FRI VL FR2 VL FR3 VL FR4 VL(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) L0EIVLTQSPGTLSLSPGER WYQQKPGQAPRLLIY GIPDRFSGSGSGTDFTLTISRL FGQGTKVEIKATLSC (129) (132) EPEDFAVYYC (136) (139) L1 EIVLTQSPGTLSLSPGERWYQQKPGQAPRLLIY GIPDRFSGSGSGTSYTLTISRL FGQGTKVEIK ATLSC (129) (132)EPEDFAVYYC (137) (139) L2 EILLTQSPGTLSLSPGER WFQQKPGQAPRLLIYGIPDRFSGSGSGTSYTLTISRL FGQGTKVEIK ATLSC (130) (133) EPEDFAVYYC (137)(139) L3 EILLTQSPGTLSLSPGER WFQQKPGQAPRLWIY GVPDRFSGSGSGTSYTLTISRLFGQGTKVEIK ATLSC (130) (134) EPEDFAVYYC (138) (139) L4EILLTQSPGTLSLSPGER WFQQKPGQSPRLWIY GVPDRFSGSGSGTSYTLTISRL FGQGTKVEIKATLTC (131) (135) EPEDFAVYYC (138) (139) *The light chain frameworkregions described in Table 5 are determined based upon the boundaries ofthe Kabat numbering system for CDRs. In other words, the VL CDRs aredetermined by Kabat and the framework regions are the amino acidresidues surrounding the CDRs in the variable region in the format FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4.

TABLE 6 Exemplary murine anti-LAG-3 antibodies. Heavy chain variableLight chain variable Antibody region SEQ ID NO: region SEQ ID NO: P01C091 2 P05E01 3 4 P01A12 5 6 P13B01 7 8 P13C10 9 10 P13C08 11 12 P13E02 1310 P13F02 14 10 P13B02 15 16 P13A06 17 18 P14C04 19 8 P14A04 20 21P15F06 22 23 P13B03 24 10 P15C02 9 25 P16D04 9 26 P13F01 20 2 P13A04 2728 P16H05 29 30 P13F09 31 32 P13G01 33 34 P13H05 35 10 P13D04 36 37P14G01 38 21 P14G03 39 40 P13G05 9 2 P13F06 9 41 P13B11 42 43 P14F01 944 P14F06 45 32 P13D05 46 47 P13G04 48 10 P15E06 20 49 P15G05 33 50P15B06 51 52 P14B07 53 8 P05E03 38 2 P13C06 54 55

TABLE 7 Exemplary humanized anti-LAG-3 antibodies. * Heavy chainvariable Light chain variable Antibody region (SEQ ID NO:) region (SEQID NO:) P13B02-01 H0 (56) L0 (73) P13B02-02 H0 (56) L1 (74) P13B02-03 H0(56) L2 (75) P13B02-04 H0 (56) L3 (76) P13B02-05 H0 (56) L4 (77)P13B02-06 H1 (57) L0 (73) P13B02-07 H1 (57) L1 (74) P13B02-08 H1 (57) L2(75) P13B02-09 H1 (57) L3 (76) P13B02-10 H1 (57) L4 (77) P13B02-11 H2(58) L0 (73) P13B02-12 H2 (58) L1 (74) P13B02-13 H2 (58) L2 (75)P13B02-14 H2 (58) L3 (76) P13B02-15 H2 (58) L4 (77) P13B02-16 H3 (59) L0(73) P13B02-17 H3 (59) L1 (74) P13B02-18 H3 (59) L2 (75) P13B02-19 H3(59) L3 (76) P13B02-20 H3 (59) L4 (77) P13B02-21 H4 (60) L0 (73)P13B02-22 H4 (60) L1 (74) P13B02-23 H4 (60) L2 (75) P13B02-24 H4 (60) L3(76) P13B02-25 H4 (60) L4 (77) P13B02-26 H4_R98K (61) L4 (77) P13B02-27H4_D100E (62) L4 (77) P13B02-28 H1_R98K (63) L0 (73) P13B02-29H1_R98K_K23T (64) L0 (73) P13B02-30 H1_R98K_L4M (65) L0 (73) P13B02-31H1_R98K_L4M_K23T (66) L0 (73) P13B02-32 H1_R98K_L4M_V5K (67) L0 (73)P13B02-33 H1_R98K_L4M_V5K_K23T (68) L0 (73) P13B02-34 H1_R98K_V5K (69)L0 (73) P13B02-35 H1_R98K_V5K_K23T (70) L0 (73) P13B02-36 H2_R98K (71)L0 (73) P13B02-37 H3_R98K (72) L0 (73)

TABLE 8 Human germline sequences. SEQ ID NO: DescriptionAmino acid sequence 153 IGHV1-46*01 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS GGSTSYAQKFQGRVTMTRDTSISTVYMELSSLRSEDTAVYYCAR 154 IGHV1-69-2*01  EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPE DGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAT 155 IGHV1-3*01  QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAG NGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR 156 IGHV1-24*01 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPE DGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT 157 IGHV1-2*01  QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPN SGGTNYAQKFQGRVTSTRDTSISTAYMELSRLRSDDTVVYYCAR 158 IGHV1-45*01 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYRYLHWVRQAPGQALEWMGWITPF NGNTNYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR 159 IGHV1-18*01 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY NGNTNYAQKLQGRVTMTTDTSISTAYMELRSLRSDDTAVYYCAR 200 IGHJ1*01  AEYFQHWGQGTLVTVSS 160 IGKV3-20*01 EIVLTQSPGTLSLSPGERATLSCRASQ SVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEP EDFAVYYCQQYGSSP 161 IGKV3D-15*01EIVMTQSPATLSVSPGERATLSCRASQ or SVSSNLAWYQQKPGQAPRLLIYGASTR IGKV3-15*01ATGIPARFSGSGSGTEFTLTISSLQSE DFAVYYCQQYNNWP 162 IGKV3D-20*01EIVLTQSPATLSLSPGERATLSCGASQ SVSSSYLAWYQQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEP EDFAVYYCQQYGSSP 163 IGKV3D-7*01 EIVMTQSPATLSLSPGERATLSCRASQ SVSSSYLSWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQP EDFAVYYCQQDYNLPP 164 IGKV1-9*01DIQLTQSPSFLSASVGDRVTITCRASQ GISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQLNSYP 165 IGKV3-11*01EIVLTQSPATLSLSPGERATLSCRASQ SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPE DFAVYYCQQRSNWP 201 IGKE*01 WTFGQGTKVEIK

TABLE 9 Exemplary sequences of LAG-3. SEQ ID NO: DescriptionAmino acid Sequence 166 Human LAG-3 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWimmature  AQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQ proteinPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY  (P18627-1)TVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDF SLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPA SVHWFRNRGQGRVPVRESPHEIFILAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGL EPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQA GTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFS GPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLS LLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL 167 Human LAG-3VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGV matureTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGP proteinRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGR QRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSR PDRPASVHWFRNRGQGRVPVRESPHREILAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIIVIYN LTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLED VSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTP SQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFL ILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEP EPEQL 210 Human LAG-3LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSL matureLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAA proteinPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQ LDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVIL NCSFSRPDRPASVHWFRNRGQGRVPVRESPHREILAESFLFLPQVSPMDSGPWGCILTYRDGFNVS IIVIYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDF TLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVW SSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAG HLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEP EPEPEPEPEQL 217 Human LAG-3LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSL fragmentLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAA  PSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLR DRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHRH LAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIIVIYNLTVLGLEPPTPLTVYAGAGSRVGLPCRL PAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAI ITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQ GERLLGAAVYFTELSSPGAQRSGRAPGALPAGH L199 Human LAG-3 GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY 30 amino acid loop 211LAG-3 epitope PTIPLQD 212 LAG-3 epitope SPTIPLQD 213 LAG-3 epitopeSPTIPLQDL 214 LAG-3 epitope SPTIPLQDLS 215 LAG-3 epitope SPTIPLQDLSL 216LAG-3 epitope SPTIPLQDLSLL

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a VH domain comprising one, two, or all three of theCDRs of a VH domain set forth in Tables 1, 2, 6, and 7 herein. Incertain embodiments, the antibody comprises the CDRH1 of one of the VHdomains set forth in Tables 1, 2, 6, and 7. In certain embodiments, theantibody comprises the CDRH2 of one of the VH domains set forth inTables 1, 2, 6, and 7. In certain embodiments, the antibody comprisesthe CDRH3 of one of the VH domains set forth in Tables 1, 2, 6, and 7.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a VL domain comprising one, two, or all three of theCDRs of a VL domain disclosed in Tables 1, 3, 6, and 7 herein. Incertain embodiments, the antibody comprises the CDRL1 of one of the VLdomains set forth in Tables 1, 3, 6, and 7. In certain embodiments, theantibody comprises the CDRL2 of one of the VL domains set forth inTables 1, 3, 6, and 7. In certain embodiments, the antibody comprisesthe CDRL3 of one of the VL domains set forth in Tables 1, 3, 6, and 7.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991),each of which is herein incorporated by reference in its entirety.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising the Kabat VH CDRs of a VH disclosed in Tables 1, 6,and 7 herein. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to LAG-3 (e.g., human LAG-3),the antibody comprising the Kabat VL CDRs of a VL disclosed in Tables 1,6, and 7 herein. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising the Kabat VH CDRs and Kabat VL CDRs ofan antibody disclosed in Tables 1, 6, and 7 herein.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226, all of which are herein incorporated by reference in theirentireties). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising the Chothia VH CDRs of a VH disclosed in Tables 1,6, and 7 herein. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising the Chothia VL CDRs of a VL disclosed inTables 1, 6, and 7 herein. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising the Chothia VH CDRsand Chothia VL CDRs of an antibody disclosed in Tables 1, 6, and 7herein. In certain embodiments, antibodies that specifically bind toLAG-3 (e.g., human LAG-3) comprise one or more CDRs, in which theChothia and Kabat CDRs have the same amino acid sequence. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3) and comprisescombinations of Kabat CDRs and Chothia CDRs.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212, each of which is herein incorporated byreference in its entirety. According to the IMGT numbering scheme, CDRH1is at positions 26 to 35, CDRH2 is at positions 51 to 57, CDRH3 is atpositions 93 to 102, CDRL1 is at positions 27 to 32, CDRL2 is atpositions 50 to 52, and CDRL3 is at positions 89 to 97.

In certain embodiments, the instant disclosure provides antibodies thatspecifically bind to LAG-3 (e.g., human LAG-3) and comprise CDRs of anantibody disclosed in Tables 1, 6, and 7 herein, as determined by theIMGT numbering system, for example, as described in Lefranc M-P (1999)supra and Lefranc M-P et al., (1999) supra.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.). In a particularembodiment, the instant disclosure provides antibodies that specificallybind to LAG-3 (e.g., human LAG-3) and comprise CDRs of an antibodydisclosed in Tables 1, 6, and 7 herein as determined by the AbMnumbering scheme.

In certain embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al., (1996) J Mol Biol 262: 732-745, whichis herein incorporated by reference in its entirety. See also, e.g.,Martin A. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001), which is hereinincorporated by reference in its entirety. In a particular embodiment,the instant disclosure provides antibodies that specifically bind toLAG-3 (e.g., human LAG-3) and comprise CDRs of an antibody disclosed inTables 1, 6, and 7 herein as described in MacCallum R M et al., (1996)supra.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a heavy chain variable region comprising theCDRH1, CDRH2, and CDRH3 region amino acid sequences of a VH domain setforth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 20, 22, 24,27, 29, 31, 33, 35, 36, 38, 39, 42, 45, 46, 48, 51, 53, 54, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72, and a lightchain variable region comprising the CDRL1, CDRL2, and CDRL3 regionamino acid sequences of a VL domain set forth in SEQ ID NO: 2, 4, 6, 8,10, 12, 16, 18, 21, 23, 25, 26, 28, 30, 32, 34, 37, 40, 41, 43, 44, 47,49, 50, 52, 55, 73, 74, 75, 76, or 77, wherein each CDR is defined inaccordance with the Kabat definition, the Chothia definition, thecombination of the Kabat definition and the Chothia definition, the IMGTnumbering system, the AbM definition, or the MacCallum definition ofCDR.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising:

(a) a CDRH1 comprises the amino acid sequence of DX₁YX₂X₃ (SEQ ID NO:140), wherein

-   -   X₁ is T or N,    -   X₂ is I or M, and    -   X₃ is H, Y or D; and/or        (b) a CDRH2 comprises the amino acid sequence of        X₁IDPANX₂X₃X₄X₅X₆X₇PX₈X₉QX₁₀ (SEQ ID NO: 142), wherein    -   X₁ is E, R, S, or K,    -   X₂ is D or G,    -   X₃ is N or H,    -   X₄ is T or 5,    -   X₅ is K or H,    -   X₆ is Y or F,    -   X₇ is D or A,    -   X₈ is K or R,    -   X₉ is F or L, and    -   X₁₀ is G or D; and/or        (c) a CDRH3 comprises the amino acid sequence of        YX₁X₂X₃YX₄VGGX₅DY (SEQ ID NO: 144), wherein    -   X₁ is Y, F, or 5,    -   X₂ is Y or D,    -   X₃ is K or R,    -   X₄ is D or E, and    -   X₅ is F or C; and/or        (d) a CDRL1 comprises the amino acid sequence of SVSSX₁ISSSX₂LX₃        (SEQ ID NO: 147), wherein    -   X₁ is S or G,    -   X₂ is N or T, and    -   X₃ is H or Y; and/or        (e) a CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID        NO: 104); and/or        (f) a CDRL3 comprises the amino acid sequence of QQWX₁X₂YPX₃T        (SEQ ID NO: 149), wherein    -   X₁ is S, N, or R,    -   X₂ is S, T or R, and    -   X₃ is F, L, H, or W.

In certain embodiments, CDRH1 comprises the amino acid sequence ofDX₁YX₂X₃ (SEQ ID NO: 141), wherein: X₁ is T or N; X₂ is I or M; and X₃is H or Y. In certain embodiments, CDRH2 comprises the amino acidsequence of X₁IDPANX₂X₃X₄KX₅X₆PX₇FQX₈ (SEQ ID NO: 143), wherein: X₁ isE, R, or S; X₂ is D or G; X₃ is N or H; X₄ is T or S; X₅ is Y or F; X₆is D or A; X₇ is K or R; and X₈ is G or D. In certain embodiments, CDRH3comprises the amino acid sequence of YX₁X₂X₃YDVGGX₄DY (SEQ ID NO: 145),wherein: X₁ is Y, F, or S; X₂ is Y or D; X₃ is K or R; and X₄ is F or C.In certain embodiments, CDRH3 comprises the amino acid sequence ofYYYX₁YX₂VGGFDY (SEQ ID NO: 146), wherein: X₁ is K or R; and X₂ is D orE. In certain embodiments, CDRL1 comprises the amino acid sequence ofSVSSSISSSNLX₁ (SEQ ID NO: 148), wherein: X₁ is H or Y. In certainembodiments, CDRL3 comprises the amino acid sequence of QQWX₁SYPX₂T (SEQID NO: 150), wherein: X₁ is S, N, or R; and X₂ is F, L, or H.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising:

(a) a CDRH1 comprises the amino acid sequence of DTYIH (SEQ ID NO: 79);and/or

(b) a CDRH2 comprises the amino acid sequence of EIDPANDNTKYDPKFQG (SEQID NO: 90); and/or

(c) a CDRH3 comprises the amino acid sequence of YYYX₁YX₂VGGFDY (SEQ IDNO: 146), wherein: X₁ is K or R; and X₂ is D or E; and/or

(d) a CDRL1 comprises the amino acid sequence of SVSSSISSSNLH (SEQ IDNO: 100); and/or

(e) a CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO:104); and/or

(f) a CDRL3 comprises the amino acid sequence of QQWSSYPFT (SEQ ID NO:105).

In certain embodiments, CDRH1 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 78-82. In certain embodiments,CDRH2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 83-93. In certain embodiments, CDRH3 comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:94-99. In certain embodiments, CDRL1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 100-103. In certainembodiments, CDRL3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 105-112.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a VH domain comprising the CDRH1, CDRH2 and CDRH3amino acid sequences set forth in SEQ ID NOs: 78, 83, and 94; 78, 85,and 95; 78, 86, and 96; 78, 86, and 97; 78, 91, and 94; 78, 92, and 96;79, 84, and 95; 79, 88, and 95; 79, 89, and 95; 79, 90, and 95; 79, 90,and 98; 79, 90, and 99; 80, 85, and 96; 81, 87, and 96; or, 82, 93, and95, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to LAG-3 (e.g.,human LAG-3), wherein the antibody comprises a VH domain comprising theCDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 79,90, and 95, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to LAG-3 (e.g.,human LAG-3), wherein the antibody comprises a VH domain comprising theCDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 79,90, and 98, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a VL domain comprising the CDRL1, CDRL2 and CDRL3amino acid sequences set forth in SEQ ID NOs: 100, 104, and 105; 100,104, and 106; 100, 104, and 107; 100, 104, and 109; 100, 104, and 110;101, 104, and 108; 102, 104, and 105; 102, 104, and 112; or, 103, 104,and 111, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to LAG-3 (e.g.,human LAG-3), wherein the antibody comprises a VL domain comprising theCDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs:100, 104, and 105, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 78, 83, 94, 100, 104, and 105; 78, 85, 95, 100, 104, and105; 78, 86, 96, 100, 104, and 105; 78, 86, 96, 100, 104, and 109; 78,86, 96, 100, 104, and 110; 78, 86, 96, 101, 104, and 108; 78, 86, 96,103, 104, and 111; 78, 86, 97, 102, 104, and 112; 78, 91, 94, 100, 104,and 107; 78, 92, 96, 100, 104, and 105; 78, 92, 96, 100, 104, and 109;79, 84, 95, 100, 104, and 105; 79, 84, 95, 100, 104, and 106; 79, 84,95, 102, 104, and 105; 79, 88, 95, 100, 104, and 105; 79, 89, 95, 100,104, and 105; 79, 90, 95, 100, 104, and 105; 79, 90, 98, 100, 104, and105; 79, 90, 99, 100, 104, and 105; 80, 85, 96, 100, 104, and 105; 81,87, 96, 100, 104, and 105; 81, 87, 96, 100, 104, and 107; or, 82, 93,95, 100, 104, and 105, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chainvariable region comprising CDRH1, CDRH2, and CDRH3 regions, and a lightchain variable region comprising CDRL1, CDRL2, and CDRL3 regions,wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 95,100, 104, and 105, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chainvariable region comprising CDRH1, CDRH2, and CDRH3 regions, and a lightchain variable region comprising CDRL1, CDRL2, and CDRL3 regions,wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 98,100, 104, and 105, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a heavy chain variable region (VH) comprisingone, two or all three of the VH CDRs of an antibody in Tables 1, 2, 6,and 7. In some embodiments, the antibody comprises one, two, three orall four of the VH framework regions described herein. In specificembodiments, the antibody comprises one, two, three or all four of theVH framework regions (FRs) set forth in Table 4 (e.g., one, two, three,or four of the framework regions in one row in Table 4). In certainembodiments, the antibody comprises one, two, three or all four of theframework regions of the heavy chain variable region sequence of SEQ IDNO: 151 or 222. In certain embodiments, the antibody comprises one, two,three or all four of the framework regions of the heavy chain variableregion sequence of SEQ ID NO: 218 or 223. In certain embodiments, theantibody comprises one, two, three or four of the framework regions of aheavy chain variable region sequence which is at least 75%, 80%, 85%,90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to one, two, three or four of theframework regions of a heavy chain variable region sequence selectedfrom the group consisting of SEQ ID NOs: 56-72 and 220. In certainembodiments, the antibody comprises a heavy chain variable frameworkregion that is or is derived from an amino acid sequence encoded by ahuman gene, wherein the amino acid sequence is selected from the groupconsisting of IGHV1-46 (e.g., IGHV1-46*01, e.g., having the amino acidsequence of SEQ ID NO: 153), IGHV1-69-2 (e.g., IGHV1-69-2*01, e.g.,having the amino acid sequence of SEQ ID NO: 154), IGHV1-3 (e.g.,IGHV1-3*01, e.g., having the amino acid sequence of SEQ ID NO: 155),IGHV1-24 (e.g., IGHV1-24*01, e.g., having the amino acid sequence of SEQID NO: 156), IGHV1-2 (e.g., IGHV1-2*01, e.g., having the amino acidsequence of SEQ ID NO: 157), IGHV1-45 (e.g., IGHV1-45*01, e.g., havingthe amino acid sequence of SEQ ID NO: 158), and IGHV1-18 (e.g.,IGHV1-18*01, e.g., having the amino acid sequence of SEQ ID NO: 159). Inspecific embodiments, the heavy chain variable framework region that isderived from said amino acid sequence consists of said amino acidsequence but for the presence of up to 20 amino acid substitutions,deletions, and/or insertions, preferably up to 20 amino acidsubstitutions. In a particular embodiment, the heavy chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues being substitutedfor an amino acid found in an analogous position in a correspondingnon-human heavy chain variable framework region. In some embodiments,the antibody comprises a heavy chain variable framework region that isderived from the amino acid sequence of SEQ ID NO: 153), wherein atleast one amino acid in the amino acid sequence of SEQ ID NO: 153 issubstituted with an amino acid in an analogous position in acorresponding non-human heavy chain variable framework region. In aspecific embodiment, the amino acid substitution is at an amino acidposition selected from the group consisting of 4, 5, 12, 23, 27, 28, 29,30, 48, 69, 71, 75, 76, 80, 81, and 94, wherein the amino acid positionis indicated according to the Kabat numbering system. In particularembodiments, the amino acid substitution is selected from the groupconsisting of 4M, 5K, 12V, 23T, 27F, 28N, 291, 30K, 48I, 69I, 71A, 75S,76N, 80L, 81Q, and 94T, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system. Inanother specific embodiment, the amino acid substitution is at an aminoacid position selected from the group consisting of 4, 27, 28, 29, 30,69, 71, and 94, wherein the amino acid position is indicated accordingto the Kabat numbering system. In particular embodiments, the amino acidsubstitution is selected from the group consisting of 4M, 27F, 28N, 291,30K, 691, 71A, and 94T, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a light chain variable region (VH) comprisingone, two or all three of the VL CDRs of an antibody in Tables 1, 3, 6,and 7. In some embodiments, the antibody comprises one, two, three orall four of the VL framework regions described herein. In specificembodiments, the antibody comprises one, two, three or all four of theVL framework regions (FRs) set forth in Table 5 (e.g., one, two, three,or four of the framework regions in one row in Table 5). In certainembodiments, the antibody comprises one, two, three or all four of theframework regions of the light chain variable region sequence of SEQ IDNO: 152 or 224. In certain embodiments, the antibody comprises one, two,three or four of the framework regions of a light chain variable regionsequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to one, two, three or four of the framework regions of a lightchain variable region sequence selected from the group consisting of SEQID NOs: 73-77. In certain embodiments, the antibody comprises a lightchain variable framework region that is or is derived from an amino acidsequence encoded by a human gene, wherein the amino acid sequence isselected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g.,having the amino acid sequence of SEQ ID NO: 160), IGKV3D-15 (e.g.,IGKV3D-15*01, e.g., having the amino acid sequence of SEQ ID NO: 161),IGKV3-15 (e.g., IGKV3-15*01, e.g., having the amino acid sequence of SEQID NO: 161), IGKV3D-20 (e.g., IGKV3D-20*01, e.g., having the amino acidsequence of SEQ ID NO: 162), IGKV3D-7 (e.g., IGKV3D-7*01, e.g., havingthe amino acid sequence of SEQ ID NO: 163), IGKV1-9 (e.g., IGKV1-9*01,e.g., having the amino acid sequence of SEQ ID NO: 164), and IGKV3-11(e.g., IGKV3-11*01, e.g., having the amino acid sequence of SEQ ID NO:165). In specific embodiments, the light chain variable framework regionthat is derived from said amino acid sequence consists of said aminoacid sequence but for the presence of up to 20 amino acid substitutions,deletions, and/or insertions, preferably up to 20 amino acidsubstitutions. In a particular embodiment, the light chain variableframework region that is derived from said amino acid sequence consistsof said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues being substitutedfor an amino acid found in an analogous position in a correspondingnon-human light chain variable framework region. In some embodiments,the antibody comprises a light chain variable framework region that isderived from the amino acid sequence of SEQ ID NO: 160, wherein at leastone amino acid in the amino acid sequence of SEQ ID NO: 160 issubstituted with an amino acid in an analogous position in acorresponding non-human light chain variable framework region. In aspecific embodiment, the amino acid substitution is at an amino acidposition selected from the group consisting of 3, 22, 36, 43, 47, 58,70, and 71, wherein the amino acid position is indicated according tothe Kabat numbering system. In particular embodiments, the amino acidsubstitution is selected from the group consisting of 3L, 22T, 36F, 43S,47W, 58V, 70S, and 71Y, wherein the position of the amino acidsubstitution is indicated according to the Kabat numbering system.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a heavy chain variable region (VH) comprisingone, two or all three of the VH CDRs of an antibody in Tables 1, 2, 6,and 7 (e.g., the VH CDRs in one row of Table 2) and a light chainvariable region (VL) comprising one, two or all three of the VL CDRs ofan antibody in Tables 1, 3, 6, and 7 (e.g., the VL CDRs in one row ofTable 3). In some embodiments, the antibody comprises the VH frameworkregions and the VL framework regions described herein. In specificembodiments, the antibody comprises the VH framework regions (FRs) setforth in Table 4 (e.g., one, two, three, or four of the frameworkregions in one row in Table 4) and the VL framework regions (FRs) setforth in Table 5 (e.g., one, two, three, or four of the frameworkregions in one row in Table 5).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a VH domain comprising the CDRH1, CDRH2 and CDRH3amino acid sequences set forth in SEQ ID NOs: 79, 90, and 95; or 79, 90,and 98, respectively. In certain embodiments, the antibody comprisesone, two, three or all four VH framework regions derived from the VH ofa human or primate antibody. In some embodiments, the antibody comprisesVH framework regions of an antibody set forth in Table 4.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a VL domain comprising the CDRL1, CDRL2 and CDRL3amino acid sequences set forth in SEQ ID NOs: 100, 104, and 105,respectively. In certain embodiments, the antibody comprises one, two,three or all four VL framework regions derived from the VL of a human orprimate antibody. In some embodiments, the antibody comprises VLframework regions of an antibody set forth in Table 5.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 79, 90, 95, 100, 104, and 105; or 79, 90, 98, 100, 104, and105, respectively. In certain embodiments, the antibody comprises one,two, three or all four VH framework regions derived from the VH of ahuman or primate antibody and one, two, three or all four VL frameworkregions derived from the VL of a human or primate antibody. In someembodiments, the antibody comprises VH framework regions and VLframework regions of an antibody set forth in Tables 4 and 5,respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 151 or 222. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), comprising a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 218 or 223. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), comprising a heavychain variable region comprising an amino acid sequence that is at least75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequenceset forth in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66,67, 68, 69, 70, 71, or 72. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66,67, 68, 69, 70, 71, or 72, optionally wherein the amino acid residue atposition 1 of the heavy chain variable region has been converted topyroglutamate. In certain embodiments, the antibody comprises a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 65. In certain embodiments, the antibody comprises a heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:220. In certain embodiments, the X in SEQ ID NO: 220 is Q. In certainembodiments, the X in SEQ ID NO: 220 is pyroglutamate. In certainembodiments, the antibody comprises a heavy chain having the amino acidsequence set forth in SEQ ID NO: 168, 225, 169, 226, 170, 227, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, or 186,optionally wherein the amino acid residue at position 1 of the heavychain has been converted to pyroglutamate. In certain embodiments, theantibody comprises a heavy chain having the amino acid sequence setforth in SEQ ID NO: 168. In certain embodiments, the antibody comprisesa heavy chain having the amino acid sequence set forth in SEQ ID NO:225. In certain embodiments, the X in SEQ ID NO: 225 is Q. In certainembodiments, the X in SEQ ID NO: 225 is pyroglutamate. In certainembodiments, the antibody comprises a heavy chain having the amino acidsequence set forth in SEQ ID NO: 169. In certain embodiments, theantibody comprises a heavy chain having the amino acid sequence setforth in SEQ ID NO: 226. In certain embodiments, the X in SEQ ID NO: 226is Q. In certain embodiments, the X in SEQ ID NO: 226 is pyroglutamate.In certain embodiments, the antibody comprises a heavy chain having theamino acid sequence set forth in SEQ ID NO: 170. In certain embodiments,the antibody comprises a heavy chain having the amino acid sequence setforth in SEQ ID NO: 227. In certain embodiments, the X in SEQ ID NO: 227is Q. In certain embodiments, the X in SEQ ID NO: 227 is pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3),comprising a light chain variable region comprising an amino acidsequence of SEQ ID NO: 152 or 224. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), comprising a light chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%) identical to the amino acid sequence set forth in SEQ IDNO: 73, 74, 75, 76, or 77. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 73, 74, 75, 76, or 77, optionally wherein theamino acid residue at position 1 of the light chain variable region hasbeen converted to pyroglutamate. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 73. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 221. In certain embodiments, the X in SEQ ID NO:221 is E. In certain embodiments, the X in SEQ ID NO: 221 ispyroglutamate. In certain embodiments, the antibody comprises a lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 187, 188, 189, 190, or 191, optionally wherein the amino acidresidue at position 1 of the light chain has been converted topyroglutamate. In certain embodiments, the antibody comprises a lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 187. In certain embodiments, the antibody comprises a light chainvariable region having the amino acid sequence set forth in SEQ ID NO:228. In certain embodiments, the X in SEQ ID NO: 228 is E. In certainembodiments, the X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 151 or 222, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 152 or 224. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), comprising a heavychain variable region comprising an amino acid sequence of SEQ ID NO:218 or 223, and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 152 or 224. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%) identical to the amino acid sequence set forth in SEQ IDNO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66, 67, 68, 69, 70, 71,or 72, and a light chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 73, 221,74, 75, 76, or 77. In certain embodiments, the antibody comprises aheavy chain variable region having the amino acid sequence set forth inSEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66, 67, 68, 69,70, 71, or 72, and a light chain variable region having the amino acidsequence set forth in SEQ ID NO: 73, 221, 74, 75, 76, or 77. In certainembodiments, the antibody comprises a heavy chain variable region andlight chain variable region having the amino acid sequences set forth inSEQ ID NO: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73;71 and 73; or 72 and 73, respectively. In certain embodiments, theantibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:56 and 73, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 220 and 221, respectively.In certain embodiments, the X in SEQ ID NO: 220 is Q. In certainembodiments, the X in SEQ ID NO: 220 is pyroglutamate. In certainembodiments, the X in SEQ ID NO: 221 is E. In certain embodiments, the Xin SEQ ID NO: 221 is pyroglutamate. In certain embodiments, the X in SEQID NO: 220 is Q, and the X in SEQ ID NO: 221 is E. In certainembodiments, the X in SEQ ID NO: 220 is Q, and the X in SEQ ID NO: 221is pyroglutamate. In certain embodiments, the X in SEQ ID NO: 220 ispyroglutamate, and the X in SEQ ID NO: 221 is E. In certain embodiments,the X in SEQ ID NO: 220 is pyroglutamate, and the X in SEQ ID NO: 221 ispyroglutamate. In certain embodiments, the antibody comprises a heavychain variable region and light chain variable region having the aminoacid sequences set forth in SEQ ID NO: 56 and 74, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 56 and 75, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:56 and 76, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 56 and 77, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 57 and 73, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:57 and 74, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 57 and 75, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 57 and 76, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:57 and 77, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 58 and 73, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 58 and 74, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:58 and 75, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 58 and 76, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 58 and 77, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:59 and 73, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 59 and 74, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 59 and 75, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:59 and 76, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 59 and 77, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 60 and 73, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:60 and 74, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 60 and 75, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 60 and 76, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:60 and 77, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 61 and 77, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 62 and 77, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:63 and 73, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 64 and 73, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 65 and 73, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:66 and 73, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 67 and 73, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 68 and 73, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:69 and 73, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 70 and 73, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 71 and 73, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:72 and 73, respectively. In certain embodiments, the amino acid residueat position 1 of the heavy chain variable region has been converted topyroglutamate. In certain embodiments, the amino acid residue atposition 1 of the light chain variable region has been converted topyroglutamate. In certain embodiments, the amino acid residue atposition 1 of the heavy chain variable region has been converted topyroglutamate, and the amino acid residue at position 1 of the lightchain variable region has been converted to pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that cross-competes for binding to LAG-3 (e.g., human LAG-3)with an antibody comprising the heavy and light chain variable regionamino acid sequences set forth in SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6;7 and 8; 9 and 10; 11 and 12; 13 and 10; 14 and 10; 15 and 16; 17 and18; 19 and 8; 20 and 21; 22 and 23; 24 and 10; 9 and 25; 9 and 26; 20and 2; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 10; 36 and 37;38 and 21; 39 and 40; 9 and 2; 9 and 41; 42 and 43; 9 and 44; 45 and 32;46 and 47; 48 and 10; 20 and 49; 33 and 50; 51 and 52; 53 and 8; 38 and2; or 54 and 55, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that cross-competes for bindingto LAG-3 (e.g., human LAG-3) with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73;57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221;220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71and 73; or 72 and 73, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of LAG-3(e.g., an epitope of human LAG-3) as an antibody described herein, e.g.,an antibody comprising the heavy and light chain variable region aminoacid sequences set forth in SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6; 7 and8; 9 and 10; 11 and 12; 13 and 10; 14 and 10; 15 and 16; 17 and 18; 19and 8; 20 and 21; 22 and 23; 24 and 10; 9 and 25; 9 and 26; 20 and 2; 27and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 10; 36 and 37; 38 and21; 39 and 40; 9 and 2; 9 and 41; 42 and 43; 9 and 44; 45 and 32; 46 and47; 48 and 10; 20 and 49; 33 and 50; 51 and 52; 53 and 8; 38 and 2; or54 and 55, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that binds to the same or an overlappingepitope of LAG-3 (e.g., an epitope of human LAG-3) as an antibodydescribed herein, e.g., an antibody comprising the heavy and light chainvariable region amino acid sequences set forth in SEQ ID NOs: 56 and 73;56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73; or 72 and73, respectively. In certain embodiments, the epitope of an antibody canbe determined by, e.g., NMR spectroscopy, surface plasmon resonance(BIAcore®), X-ray diffraction crystallography studies, ELISA assays,hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquidchromatography electrospray mass spectrometry), array-basedoligo-peptide scanning assays, and/or mutagenesis mapping (e.g.,site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303, all of which are herein incorporated byreference in their entireties). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323, all of which are herein incorporatedby reference in their entireties). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antibody is determined using alanine scanning mutagenesisstudies. In addition, antibodies that recognize and bind to the same oroverlapping epitopes of LAG-3 (e.g., human LAG-3) can be identifiedusing routine techniques such as an immunoassay, for example, by showingthe ability of one antibody to block the binding of another antibody toa target antigen, i.e., a competitive binding assay. Competition bindingassays also can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asLAG-3 (e.g., human LAG-3). Numerous types of competitive binding assaysare known, for example: solid phase direct or indirect radioimmunoassay(RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid phase direct biotin-avidin EIA (see Kirkland T N et al.,(1986) J Immunol 137: 3614-9); solid phase direct labeled assay, solidphase direct labeled sandwich assay (see Harlow E & Lane D, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using I-125 label (see Morel G A et al., (1988) MolImmunol 25(1): 7-15); solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled RIA (seeMoldenhauer G et al., (1990) Scand J Immunol 32: 77-82), all of whichare herein incorporated by reference in their entireties. Typically,such an assay involves the use of purified antigen (e.g., LAG-3 such ashuman LAG-3) bound to a solid surface or cells bearing either of these,an unlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition bindingassay can be configured in a large number of different formats usingeither labeled antigen or labeled antibody. In a common version of thisassay, the antigen is immobilized on a 96-well plate. The ability ofunlabeled antibodies to block the binding of labeled antibodies to theantigen is then measured using radioactive or enzyme labels. For furtherdetails see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274;Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al.,(1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane Deditors supra, pp. 386-389, all of which are herein incorporated byreference in their entireties.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 168, 225, 169, 226, 170, 227, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, or 186,optionally wherein the amino acid residue at position 1 of the heavychain has been converted to pyroglutamate. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 168. In certain embodiments, the antibody comprisesa heavy chain comprising the amino acid sequence set forth in SEQ ID NO:225. In certain embodiments, the X in SEQ ID NO: 225 is Q. In certainembodiments, the X in SEQ ID NO: 225 is pyroglutamate. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO: 169. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 226. In certain embodiments, the X in SEQ ID NO: 226is Q. In certain embodiments, the X in SEQ ID NO: 226 is pyroglutamate.In certain embodiments, the antibody comprises a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO: 170. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO: 227. In certain embodiments, the Xin SEQ ID NO: 227 is Q. In certain embodiments, the X in SEQ ID NO: 227is pyroglutamate. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 171. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 172. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 173. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 174. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 175. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 176. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 177. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 178. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 179. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 180. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 181. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 182. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 183. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 184. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 185. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 186. In certain embodiments, the amino acid residue atposition 1 of the heavy chain has been converted to pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a light chain comprising the amino acid sequence setforth in SEQ ID NO: 187, 228, 188, 189, 190, or 191, optionally whereinthe amino acid residue at position 1 of the light chain has beenconverted to pyroglutamate. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence set forth inSEQ ID NO: 187. In certain embodiments, the antibody comprises a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 228. Incertain embodiments, the X in SEQ ID NO: 228 is E. In certainembodiments, the X in SEQ ID NO: 228 is pyroglutamate. In certainembodiments, the antibody comprises a light chain comprising the aminoacid sequence set forth in SEQ ID NO: 188. In certain embodiments, theantibody comprises a light chain comprising the amino acid sequence setforth in SEQ ID NO: 189. In certain embodiments, the antibody comprisesa light chain comprising the amino acid sequence set forth in SEQ ID NO:190. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 191. Incertain embodiments, the amino acid residue at position 1 of the lightchain has been converted to pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 168; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 225; and a light chain comprising the amino acidsequence of SEQ ID NO: 228. In certain embodiments, the X in SEQ ID NO:225 is Q, and the X in SEQ ID NO: 228 is pyroglutamate. In certainembodiments, the X in SEQ ID NO: 225 is pyroglutamate, and the X in SEQID NO: 228 is E. In certain embodiments, the X in SEQ ID NO: 225 ispyroglutamate, and the X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 169; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 226; and a light chain comprising the amino acidsequence of SEQ ID NO: 228. In certain embodiments, the X in SEQ ID NO:226 is Q, and the X in SEQ ID NO: 228 is pyroglutamate. In certainembodiments, the X in SEQ ID NO: 226 is pyroglutamate, and the X in SEQID NO: 228 is E. In certain embodiments, the X in SEQ ID NO: 226 ispyroglutamate, and the X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 170; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 227; and a light chain comprising the amino acidsequence of SEQ ID NO: 228. In certain embodiments, the X in SEQ ID NO:227 is Q, and the X in SEQ ID NO: 228 is pyroglutamate. In certainembodiments, the X in SEQ ID NO: 227 is pyroglutamate, and the X in SEQID NO: 228 is E. In certain embodiments, the X in SEQ ID NO: 227 ispyroglutamate, and the X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 171; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 172; and a light chain comprising the amino acidsequence of SEQ ID NO: 187. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 173; and a light chaincomprising the amino acid sequence of SEQ ID NO: 187. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 174; anda light chain comprising the amino acid sequence of SEQ ID NO: 187. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 175; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 176; and a light chain comprising the amino acidsequence of SEQ ID NO: 187. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 177; and a light chaincomprising the amino acid sequence of SEQ ID NO: 187. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 178; anda light chain comprising the amino acid sequence of SEQ ID NO: 187. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 179; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 180; and a light chain comprising the amino acidsequence of SEQ ID NO: 187. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 181; and a light chaincomprising the amino acid sequence of SEQ ID NO: 187. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 182; anda light chain comprising the amino acid sequence of SEQ ID NO: 187. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 183; and a light chain comprising the amino acid sequence ofSEQ ID NO: 187. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to LAG-3 (e.g., humanLAG-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 184; and a light chain comprising the amino acidsequence of SEQ ID NO: 187. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 185; and a light chaincomprising the amino acid sequence of SEQ ID NO: 187. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 186; anda light chain comprising the amino acid sequence of SEQ ID NO: 187. Incertain embodiments, the amino acid residue at position 1 of the heavychain has been converted to pyroglutamate. In certain embodiments, theamino acid residue at position 1 of the light chain has been convertedto pyroglutamate. In certain embodiments, the amino acid residue atposition 1 of the heavy chain has been converted to pyroglutamate, andthe amino acid residue at position 1 of the light chain has beenconverted to pyroglutamate.

Any Ig constant region can be used in the antibodies disclosed herein.In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD,IgA, or IgY immunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 192, 193, 194, 195, 196, 197, 208, or 209.In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 194. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 195. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a light chain constant region comprising the aminoacid sequence of SEQ ID NO: 198. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a light chainconstant region comprising the amino acid sequence of SEQ ID NO: 219.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain may be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are hereinincorporated by reference in their entireties, for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In some embodiments, one, two or more amino acidmutations (e.g., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (e.g., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), numberedaccording to the EU numbering system. In a specific embodiment, theconstant region of the IgG₁ of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU numbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of mutant IgG,referred to as “YTE mutant” has been shown to display fourfold increasedhalf-life as compared to wild-type versions of the same antibody (seeDall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24, which is hereinincorporated by reference in its entirety). In certain embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, all of which are hereinincorporated by reference in their entireties.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU numbering system, can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, each of which is herein incorporated by reference in itsentirety. In some embodiments, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain may reduceFc receptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each ofwhich is herein incorporated by reference in its entirety, for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In certain embodiments, one ormore amino acid substitutions may be introduced into the Fc region of anantibody described herein to remove potential glycosylation sites on Fcregion, which may reduce Fc receptor binding (see, e.g., Shields R L etal., (2001) J Biol Chem 276: 6591-604, which is herein incorporated byreference in its entirety). In various embodiments, one or more of thefollowing mutations in the constant region of an antibody describedherein may be made: an N297A substitution; an N297Q substitution; aL235A substitution and a L237A substitution; a L234A substitution and aL235A substitution; a E233P substitution; a L234V substitution; a L235Asubstitution; a C236 deletion; a P238A substitution; a D265Asubstitution; a A327Q substitution; or a P329A substitution, numberedaccording to the EU numbering system. In certain embodiments, a mutationselected from the group consisting of D265A, P329A, and a combinationthereof may be made in the constant region of an antibody describedherein.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system. In oneembodiment, an antibody described herein comprises the constant domainof an IgG₁ with a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numberingsystem. In another embodiment, an antibody described herein comprisesthe constant domain of an IgG₁ with a mutation selected from the groupconsisting of L234A, L235A, and a combination thereof, numberedaccording to the EU numbering system. In certain embodiments, amino acidresidues in the constant region of an antibody described herein in thepositions corresponding to positions L234, L235, and D265 in a humanIgG₁ heavy chain, numbered according to the EU index of numbering, arenot L, L, and D, respectively. This approach is described in detail inInternational Publication No. WO 14/108483, which is herein incorporatedby reference in its entirety. In a particular embodiment, the aminoacids corresponding to positions L234, L235, and D265 in a human IgG₁heavy chain are F, E, and A; or A, A, and A, respectively.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al), which is herein incorporated byreference in its entirety. In some embodiments, one or more amino acidresidues within amino acid positions 231 to 238 in the N-terminal regionof the CH2 domain of an antibody described herein are altered to therebyalter the ability of the antibody to fix complement. This approach isdescribed further in International Publication No. WO 94/29351, which isherein incorporated by reference in its entirety. In certainembodiments, the Fc region of an antibody described herein is modifiedto increase the ability of the antibody to mediate antibody dependentcellular cytotoxicity (ADCC) and/or to increase the affinity of theantibody for an Fcγ receptor by mutating one or more amino acids (e.g.,introducing amino acid substitutions) at the following positions: 238,239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272,276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298,301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329,330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, numberedaccording to the EU numbering system. This approach is described furtherin International Publication No. WO 00/42072, which is hereinincorporated by reference in its entirety.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3), the antibody comprising a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 196. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 197.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody described herein having two heavychain constant regions.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) andfunctions as an antagonist.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) anddecreases LAG-3 (e.g., human LAG-3) activity by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein and/orknown to one of skill in the art, relative to LAG-3 (e.g., human LAG-3)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to LAG-3 (e.g., human LAG-3)).In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) anddecreases LAG-3 (e.g., human LAG-3) activity by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold as assessed by methods described herein and/or knownto one of skill in the art, relative to LAG-3 (e.g., human LAG-3)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to LAG-3 (e.g., human LAG-3)).Non-limiting examples of LAG-3 (e.g., human LAG-3) activity can includeLAG-3 (e.g., human LAG-3) signaling, LAG-3 (e.g., human LAG-3) bindingto LAG-3 (e.g., human LAG-3) ligand (e.g., MHC class II), and inhibitionof cytokine production (e.g., IL-2 and/or TNF-α). In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to LAG-3 (e.g., human LAG-3) and deactivates,reduces, or inhibits a LAG-3 (e.g., human LAG-3) activity. In specificembodiments, a decrease in a LAG-3 (e.g., human LAG-3) activity isassessed as described in the Examples, infra.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) andreduces LAG-3 (e.g., human LAG-3) binding to its ligand (e.g., MHC classII) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art, relative to LAG-3 (e.g., human LAG-3) binding to itsligand (e.g., MHC class II) without any antibody or with an unrelatedantibody (e.g., an antibody that does not specifically bind to LAG-3(e.g., human LAG-3)). In specific embodiments, the instant disclosureprovides an isolated antibody that specifically binds to LAG-3 (e.g.,human LAG-3) and reduces LAG-3 (e.g., human LAG-3) binding to its ligand(e.g., MHC class II) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods described herein (see the Examples, infra) or knownto one of skill in the art, relative to LAG-3 (e.g., human LAG-3)binding to its ligand (e.g., MHC class II) without any antibody or withan unrelated antibody (e.g., an antibody that does not specifically bindto LAG-3 (e.g., human LAG-3)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) andincreases cytokine production (e.g., IL-2 and/or TNF-α) by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art, relative to cytokine production without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toLAG-3 (e.g., human LAG-3)). In specific embodiments, the instantdisclosure provides an isolated antibody that specifically binds toLAG-3 (e.g., human LAG-3) and increases cytokine production (e.g., IL-2and/or TNF-α) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold,2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art, relative to cytokine production without any antibodyor with an unrelated antibody (e.g., an antibody that does notspecifically bind to LAG-3 (e.g., human LAG-3)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) and eitheralone or in combination with an anti-PD-1 antibody (e.g., pembrolizumabor nivolumab), an anti-PD-L1 antibody (e.g., avelumab, durvalumab, oratezolizumab), or an anti-CTLA-4 antibody (e.g., ipilimumab) increasesIL-2 production in human peripheral blood mononuclear cells (PBMCs) inresponse to Staphylococcus Enterotoxin A (SEA) stimulation by at leastabout 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold, as assessed by methods described herein (seethe Examples, infra) or known to one of skill in the art, relative toIL-2 production without any antibody or with an unrelated antibody(e.g., an antibody that does not specifically bind to LAG-3 (e.g., humanLAG-3)).

In certain embodiments, human peripheral blood mononuclear cells (PBMCs)stimulated with Staphylococcus Enterotoxin A (SEA) in the presence of anantibody described herein, which specifically binds to LAG-3 (e.g.,human LAG-3), have increased IL-2 production by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold relative to PBMCs only stimulated with SEA without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to LAG-3 (e.g., human LAG-3)), as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) and eitheralone or in combination with an anti-PD-1 antibody (e.g., pembrolizumabor nivolumab) increases TNFα production in tumor infiltratinglymphocytes (TILs) in response to anti-CD3 antibody and anti-CD28antibody stimulation by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods described herein (see the Examples, infra) or knownto one of skill in the art, relative to TNFα production without anantibody that specifically binds to LAG-3 (e.g., human LAG-3). In oneembodiment, the TILs are from renal cell carcinoma tumor. In anotherembodiment, the TILs are from colorectal cancer tumor.

In certain embodiments, tumor infiltrating lymphocytes (TILs) stimulatedwith anti-CD3 and anti-CD28 antibodies in the presence of an antibodydescribed herein, which specifically binds to LAG-3 (e.g., human LAG-3),have increased TNFα production by at least about 1.2 fold, 1.3 fold, 1.4fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 foldrelative to TILs only stimulated with anti-CD3 and anti-CD28 antibodieswithout an antibody that specifically binds to LAG-3 (e.g., humanLAG-3), as assessed by methods described herein (see the Examples,infra) or known to one of skill in the art. In one embodiment, the TILsare from renal cell carcinoma tumor. In another embodiment, the TILs arefrom colorectal cancer tumor.

6.3 Pharmaceutical Compositions

Provided herein are compositions (e.g., pharmaceutical compositions)comprising an anti-LAG-3 (e.g., human LAG-3) antibody described hereinhaving the desired degree of purity in a physiologically acceptablecarrier, excipient or stabilizer (Remington's Pharmaceutical Sciences(1990) Mack Publishing Co., Easton, Pa.). Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In certain embodiments, the composition comprises one or more anti-LAG-3(e.g., human LAG-3) antibodies as disclosed herein, wherein in a portionof the antibodies, the N-terminal amino acid residue(s) of the heavychain and/or the light chain have been converted to pyroglutamate (e.g.,as a result of post-translational cyclization of the free amino group ofthe N-terminal E or Q residue). In certain embodiments, the N-terminalamino acid residue of at least 50% (e.g., at least 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the heavy chains in thecomposition has been converted to pyroglutamate. In certain embodiments,the N-terminal amino acid residue of no more than 50% (e.g., no morethan 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, or 40%) of the lightchains in the composition has been converted to pyroglutamate. Incertain embodiments, the N-terminal amino acid residue of at least 50%(e.g., at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%) of the heavy chains in the composition has been converted topyroglutamate, and the N-terminal amino acid residue of no more than 50%(e.g., no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, or 40%)of the light chains in the composition has been converted topyroglutamate.

In a specific embodiment, pharmaceutical compositions comprise ananti-LAG-3 (e.g., human LAG-3) antibody described herein, and optionallyone or more additional prophylactic or therapeutic agents, in apharmaceutically acceptable carrier. In a specific embodiment,pharmaceutical compositions comprise an effective amount of an antibodydescribed herein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In someembodiments, the antibody is the only active ingredient included in thepharmaceutical composition. Pharmaceutical compositions described hereincan be useful in inhibiting LAG-3 (e.g., human LAG-3) activity andtreating a condition, such as cancer or an infectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-LAG-3 (e.g., human LAG-3) antibody described herein can beformulated as an aerosol for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma and are incorporated byreference in their entireties). These formulations for administration tothe respiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microtine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

An anti-LAG-3 (e.g., human LAG-3) antibody described herein can beformulated for local or topical application, such as for topicalapplication to the skin and mucous membranes, such as in the eye, in theform of gels, creams, and lotions and for application to the eye or forintracisternal or intraspinal application. Topical administration iscontemplated for transdermal delivery and also for administration to theeyes or mucosa, or for inhalation therapies. Nasal solutions of theantibody alone or in combination with other pharmaceutically acceptableexcipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, a pharmaceutical composition comprising anantibody described herein is a lyophilized powder, which can bereconstituted for administration as solutions, emulsions and othermixtures. It may also be reconstituted and formulated as solids or gels.The lyophilized powder is prepared by dissolving an antibody describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In some embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-LAG-3 (e.g., human LAG-3) antibodies described herein and othercompositions provided herein can also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874, all of which are herein incorporatedby reference in their entireties. In a specific embodiment, an antibodydescribed herein is targeted to a tumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

6.4 Methods of Use and Uses

In another aspect, the instant disclosure provides a method of treatinga subject using the anti-LAG-3 (e.g., human LAG-3) antibodies disclosedherein. Any disease or disorder in a subject that would benefit frominhibition of LAG-3 (e.g., human LAG-3) function can be treated usingthe anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein. Theanti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein areparticularly useful for inhibiting immune system tolerance to tumors,and accordingly can be used as an immunotherapy for subjects withcancer. For example, in certain embodiments, the instant disclosureprovides a method of increasing T cell activation in response to anantigen in a subject, the method comprising administering to the subjectan effective amount of an anti-LAG-3 (e.g., human LAG-3) antibody orpharmaceutical composition thereof, as disclosed herein. In certainembodiments, the instant disclosure provides a method of treating cancerin a subject, the method comprising administering to the subject aneffective amount of the antibody or pharmaceutical composition, asdisclosed herein.

Cancers that can be treated with the anti-LAG-3 (e.g., human LAG-3)antibodies or pharmaceutical compositions disclosed herein include,without limitation, a solid tumor, a hematologic cancer, leukemia,lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer,renal transitional cell cancer, bladder cancer, Wilm's cancer, ovariancancer, pancreatic cancer, breast cancer (e.g., characterized by amutation in BRCA1 and/or BRCA2), prostate cancer, bone cancer, lungcancer (e.g., non-small cell lung cancer or small cell lung cancer),gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma,head and neck cancer, squamous cell carcinoma, multiple myeloma, renalcell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma,melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma,brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibularschwannoma, a primitive neuroectodermal tumor, medulloblastoma,astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma,choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathicmyelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer,carcinoid cancer, liver cancer, epithelial cancer, and peritonealcancer. In certain embodiments, the cancer is metastatic cancer, e.g.,of the varieties described above.

In certain embodiments, the cancer is a solid tumor, a hematologicalcancer (e.g., leukemia, lymphoma, myeloma), and a metastatic lesionthereof. In one embodiment, the cancer is a solid tumor. Examples ofsolid tumors include malignancies, e.g., sarcomas and carcinomas (e.g.,adenocarcinomas) of the various organ systems, such as those affectinglung, breast, lymphoid, gastrointestinal or colorectal, genitals andgenitourinary tract (e.g., renal, urothelial, bladder cells), pharynx,CNS (e.g., brain, neural or glial cells), skin (e.g., melanoma), headand neck (e.g., head and neck squamous cell carcinoma (HNCC)), andpancreas. For example, melanoma, colon cancers, gastric cancer, rectalcancer, renal-cell carcinoma, breast cancer (e.g., a breast cancer thatdoes not express one, two or all of estrogen receptor, progesteronereceptor, or Her2/neu, e.g., a triple negative breast cancer), livercancer, a lung cancer (e.g., a non-small cell lung cancer (NSCLC) (e.g.,a NSCLC with squamous and/or non-squamous histology) or small cell lungcancer), prostate cancer, cancer of head or neck (e.g., HPV+ squamouscell carcinoma), cancer of the small intestine and cancer of theesophagus.

In one embodiment, the cancer is a hematological cancer, for example, aleukemia, a lymphoma, or a myeloma. In one embodiment, the cancer is aleukemia, for example, acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), acute myeloblastic leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronicmyeloid leukemia (CIVIL), chronic myelomonocytic leukemia (CMML),chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In oneembodiment, the cancer is a lymphoma, for example, B cell lymphoma,diffuse large B-cell lymphoma (DLBCL), activated B-cell like (ABC)diffuse large B cell lymphoma, germinal center B cell (GCB) diffuselarge B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractorynon-Hodgkin lymphoma, recurrent follicular non-Hodgkin lymphoma, Burkittlymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In oneembodiment the cancer is a myeloma, for example, multiple myeloma.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of ananti-LAG-3 (e.g., human LAG-3) antibody or pharmaceutical compositionthereof, as disclosed herein. In one embodiment, provided herein aremethods for preventing and/or treating an infection (e.g., a viralinfection, a bacterial infection, a fungal infection, a protozoalinfection, or a parasitic infection). The infection prevented and/ortreated in accordance with the methods can be caused by an infectiousagent identified herein. In a specific embodiment, an anti-LAG-3 (e.g.,human LAG-3) antibody described herein or a composition thereof is theonly active agent administered to a subject. In some embodiments, ananti-LAG-3 (e.g., human LAG-3) antibody described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases.

Infectious diseases that can be treated and/or prevented by anti-LAG-3(e.g., human LAG-3) antibodies or pharmaceutical compositions disclosedherein are caused by infectious agents including but not limited tobacteria, parasites, fungi, protozae, and viruses. In a specificembodiment, the infectious disease treated and/or prevented byanti-LAG-3 (e.g., human LAG-3) antibodies or pharmaceutical compositionsdisclosed herein is caused by a virus. Viral diseases or viralinfections that can be prevented and/or treated in accordance with themethods described herein include, but are not limited to, those causedby hepatitis type A, hepatitis type B, hepatitis type C, influenza(e.g., influenza A or influenza B), varicella, adenovirus, herpessimplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus,polio virus, small pox, Epstein Barr virus, human immunodeficiency virustype I (HIV-I), human immunodeficiency virus type II (HIV-II), andagents of viral diseases such as viral meningitis, encephalitis, dengueor small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, leishmania, coccidiosis,trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating a disease or disorder of the nervous system in asubject, the method comprising administering to the subject an effectiveamount of an anti-LAG-3 (e.g., human LAG-3) antibody or pharmaceuticalcomposition thereof, as disclosed herein. In some embodiments, thedisease or disorder of the nervous system is a synucleinopathy. In someembodiments, the disease or disorder of the nervous system isParkinson's disease.

In certain embodiments, these methods further comprise administering anadditional therapeutic agent to the subject. In certain embodiments, theadditional therapeutic agent is a chemotherapeutic, radiotherapeutic, ora checkpoint targeting agent. In certain embodiments, thechemotherapeutic agent is a hypomethylating agent (e.g., azacitidine).In certain embodiments, the checkpoint targeting agent is selected fromthe group consisting of an antagonist anti-CTLA-4 antibody, anantagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, anantagonist anti-PD-1 antibody, an antagonist anti-TIM-3 antibody, anantagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, anagonist anti-GITR antibody, an agonist anti-OX40 antibody, an antagonistanti-TIGIT antibody, an agonist anti-CD137 antibody, an antagonistanti-VISTA antibody, an antagonist anti-CD73 antibody, and an antagonistanti-CD96 antibody.

In one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof the present invention, wherein the method further comprisesadministering an additional therapeutic agent to the subject. In oneembodiment, the present invention relates to (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent for use as a medicament. In one embodiment,the present invention relates to (a) an antibody and/or pharmaceuticalcomposition of the present invention, and (b) an additional therapeuticagent for use in a method for the treatment of cancer. In a furtherembodiment, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent. In one embodiment, the additionaltherapeutic agent is a chemotherapeutic, a radiotherapeutic, or acheckpoint targeting agent.

In certain embodiments, an anti-PD-1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-1 antibody isnivolumab, also known as BMS-936558 or MDX1106, developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody ispembrolizumab, also known as lambrolizumab or MK-3475, developed byMerck & Co. In certain embodiments, the anti-PD-1 antibody ispidilizumab, also known as CT-011, developed by CureTech. In certainembodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514,developed by Medimmune. In certain embodiments, the anti-PD-1 antibodyis PDR001 developed by Novartis Pharmaceuticals. In certain embodiments,the anti-PD-1 antibody is REGN2810 developed by RegeneronPharmaceuticals. In certain embodiments, the anti-PD-1 antibody isPF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1antibody is BGB-A317 developed by BeiGene. In certain embodiments, theanti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. Incertain embodiments, the anti-PD-1 antibody is SHR-1210 developed byHengrui.

Further non-limiting examples of anti-PD-1 antibodies that may be usedin treatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties: U.S. Pat. Nos. 6,808,710; 7,332,582;7,488,802; 8,008,449; 8,114,845; 8,168,757; 8,354,509; 8,686,119;8,735,553; 8,747,847; 8,779,105; 8,927,697; 8,993,731; 9,102,727;9,205,148; U.S. Publication No. US 2013/0202623 A1; U.S. Publication No.US 2013/0291136 A1; U.S. Publication No. US 2014/0044738 A1; U.S.Publication No. US 2014/0356363 A1; U.S. Publication No. US 2016/0075783A1; and PCT Publication No. WO 2013/033091 A1; PCT Publication No. WO2015/036394 A1; PCT Publication No. WO 2014/179664 A2; PCT PublicationNo. WO 2014/209804 A1; PCT Publication No. WO 2014/206107 A1; PCTPublication No. WO 2015/058573 A1; PCT Publication No. WO 2015/085847A1; PCT Publication No. WO 2015/200119 A1; PCT Publication No. WO2016/015685 A1; and PCT Publication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-L1 antibody isatezolizumab developed by Genentech. In certain embodiments, theanti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene andMedimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab,also known as MSB0010718C, developed by Merck Serono and Pfizer. Incertain embodiments, the anti-PD-L1 antibody is MDX-1105 developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody isAMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used intreatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties: U.S. Pat. Nos. 7,943,743; 8,168,179;8,217,149; 8,552,154; 8,779,108; 8,981,063; 9,175,082; U.S. PublicationNo. US 2010/0203056 A1; U.S. Publication No. US 2003/0232323 A1; U.S.Publication No. US 2013/0323249 A1; U.S. Publication No. US 2014/0341917A1; U.S. Publication No. US 2014/0044738 A1; U.S. Publication No. US2015/0203580 A1; U.S. Publication No. US 2015/0225483 A1; U.S.Publication No. US 2015/0346208 A1; U.S. Publication No. US 2015/0355184A1; and PCT Publication No. WO 2014/100079 A1; PCT Publication No. WO2014/022758 A1; PCT Publication No. WO 2014/055897 A2; PCT PublicationNo. WO 2015/061668 A1; PCT Publication No. WO 2015/109124 A1; PCTPublication No. WO 2015/195163 A1; PCT Publication No. WO 2016/000619A1; and PCT Publication No. WO 2016/030350 A1.

In certain embodiments, an anti-CTLA-4 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-CTLA-4 antibody isipilimumab developed by Bristol-Myers Squibb. In certain embodiments,the anti-CTLA-4 antibody is ipilimumab developed by Bristol-MyersSquibb. In certain embodiments, the anti-CTLA-4 antibody is tremelimumabdeveloped by Pfizer and Medimmune.

Non-limiting examples of anti-CTLA-4 antibodies that may be used intreatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties: U.S. Pat. Nos. 6,984,720; 7,411,057;7,034,121; 8,697,845; U.S. Publication No. US 2009/0123477 A1; U.S.Publication No. US 2014/0105914 A1; U.S. Publication No. US 2013/0267688A1; U.S. Publication No. US 2016/0145355 A1; PCT Publication No. WO2014/207064 A1; and PCT Publication No. WO 2016/015675 A1.

In certain embodiments, an anti-LAG-3 (e.g., human LAG-3) antibodydisclosed herein is administered to a subject in combination with acompound that targets an immunomodulatory enzyme(s) such as IDO(indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase).In certain embodiments, such compound is selected from the groupconsisting of epacadostat (Incyte Corp; see, e.g., WO 2010/005958 whichis herein incorporated by reference in its entirety), BMS-986205 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). In one embodiment, the compound isepacadostat. In another embodiment, the compound is BMS-986205. Inanother embodiment, the compound is indoximod. In another embodiment,the compound is NLG919. In a specific embodiment, an anti-LAG-3 (e.g.,human LAG-3) antibody disclosed herein is administered to a subject incombination with an IDO inhibitor for treating cancer. The IDO inhibitoras described herein for use in treating cancer is present in a soliddosage form of a pharmaceutical composition such as a tablet, a pill ora capsule, wherein the pharmaceutical composition includes an IDOinhibitor and a pharmaceutically acceptable excipient. As such, theantibody as described herein and the IDO inhibitor as described hereincan be administered separately, sequentially or concurrently as separatedosage forms. In one embodiment, the antibody is administeredparenterally, and the IDO inhibitor is administered orally. Inparticular embodiments, the inhibitor is selected from the groupconsisting of epacadostat (Incyte Corporation), BMS-986205 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). Epacadostat has been described in PCTPublication No. WO 2010/005958, which is herein incorporated byreference in its entirety. In one embodiment, the inhibitor isepacadostat. In another embodiment, the inhibitor is BMS-986205. Inanother embodiment, the inhibitor is indoximod. In another embodiment,the inhibitor is NLG919.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to LAG-3 (e.g., human LAG-3) incombination with an anti-PD-1 antibody (e.g., pembrolizumab ornivolumab) and an inhibitor of indoleamine-2,3-dioxygenase (IDO). Incertain embodiments, the anti-PD-1 antibody is pembrolizumab. In certainembodiments, the anti-PD-1 antibody is nivolumab. In certainembodiments, the IDO inhibitor is selected from the group consisting ofepacadostat, BMS-986205, indoximod, and NLG919. In certain embodiments,the IDO inhibitor is epacadostat. In certain embodiments, the IDOinhibitor is BMS-986205. In certain embodiments, the IDO inhibitor isindoximod.

In certain embodiments, an anti-LAG-3 (e.g., human LAG-3) antibodydisclosed herein is administered to a subject in combination withanother anti-cancer agent. Exemplary anti-cancer agents include antibodytherapeutics such as trastuzumab (Herceptin), antibodies toco-stimulatory or co-inhibitory molecules such as CTLA-4, CD137, andPD-1, and antibodies to cytokines such as IL-10 and TGF-β.

In certain embodiments, the additional therapeutic agent is an inhibitorof JAK, PI3Kdelta, BRD, PI3Kgamma, or Axl/Mer. In certain embodiments,the additional therapeutic agent is an inhibitor of JAK, including JAK1and/or JAK2. In certain embodiments, the additional therapeutic agent isan inhibitor of PI3Kdelta. In certain embodiments, the additionaltherapeutic agent is an inhibitor of BRD. In certain embodiments, theadditional therapeutic agent is an inhibitor of PI3Kgamma. In certainembodiments, the additional therapeutic agent is an inhibitor ofAxl/Mer.

Additional examples of anti-cancer agents include those that blockimmune cell migration such as antagonists to chemokine receptors,including CCR2 and CCR4, and those that augment the immune system suchas adjuvants or adoptive T cell transfer.

One or more additional immune checkpoint modulators can be used incombination with an anti-LAG-3 (e.g., human LAG-3) antibody disclosedherein for treatment of any diseases, disorders, or conditions describedherein, e.g., TAM-associated diseases, disorders, or conditions.Exemplary immune checkpoint modulators include modulators against immunecheckpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47,CD96, CD137, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM,arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA,CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, PD-1, PD-L1, and PD-L2. In someembodiments, the immune checkpoint molecule is a co-stimulatorycheckpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR,and CD137. In some embodiments, the immune checkpoint molecule is aco-inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4,BTLA, CTLA-4, IDO, KIR, PD-1, TIM-3, and VISTA. In some embodiments, ananti-LAG-3 (e.g., human LAG-3) antibody disclosed herein can be used incombination with one or more agents selected from KIR inhibitors, TIGITinhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFRbeta inhibitors.

In some embodiments, the modulator of an immune checkpoint molecule isan antagonistic anti-PD1 antibody, an antagonistic anti-PD-L1 antibody,or an antagonistic anti-CTLA-4 antibody.

In some embodiments, the modulator of an immune checkpoint molecule isan agonist of GITR, e.g., an agonistic anti-GITR antibody. In someembodiments, the agonistic anti-GITR antibody is TRX518 or MK-4166.

In some embodiments, the modulator of an immune checkpoint molecule isan agonist of OX40, e.g., an agonistic anti-OX40 antibody or OX40Lfusion protein. In some embodiments, the agonistic anti-OX40 antibody isMEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383.

An anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein can be usedin combination with one or more agents for the treatment of diseasessuch as cancer. In some embodiments, the agent is an alkylating agent, aproteasome inhibitor, a corticosteroid, or an immunomodulatory agent.Examples of an alkylating agent include cyclophosphamide (CY), melphalan(MEL), and bendamustine. In some embodiments, the proteasome inhibitoris carfilzomib. In some embodiments, the corticosteroid is dexamethasone(DEX). In some embodiments, the immunomodulatory agent is lenalidomide(LEN) or pomalidomide (POM).

In certain embodiments, an anti-LAG-3 (e.g., human LAG-3) antibodydisclosed herein is administered to a subject in combination with avaccine. The vaccine can be, e.g., a peptide vaccine, a DNA vaccine, oran RNA vaccine. In certain embodiments, the vaccine is a heat shockprotein based tumor vaccine or a heat shock protein based pathogenvaccine. In a specific embodiment, an anti-LAG-3 (e.g., human LAG-3)antibody disclosed herein is administered to a subject in combinationwith a heat shock protein based tumor-vaccine. Heat shock proteins(HSPs) are a family of highly conserved proteins found ubiquitouslyacross all species. Their expression can be powerfully induced to muchhigher levels as a result of heat shock or other forms of stress,including exposure to toxins, oxidative stress or glucose deprivation.Five families have been classified according to molecular weight:HSP-110, -90, -70, -60 and -28. HSPs deliver immunogenic peptidesthrough the cross-presentation pathway in antigen presenting cells(APCs) such as macrophages and dendritic cells (DCs), leading to T cellactivation. HSPs function as chaperone carriers of tumor-associatedantigenic peptides forming complexes able to induce tumor-specificimmunity. Upon release from dying tumor cells, the HSP-antigen complexesare taken up by antigen-presenting cells (APCs) wherein the antigens areprocessed into peptides that bind MHC class I and class II moleculesleading to the activation of anti-tumor CD8+ and CD4+ T cells. Theimmunity elicited by HSP complexes derived from tumor preparations isspecifically directed against the unique antigenic peptide repertoireexpressed by the cancer of each subject. Therefore, in one embodiment,the present invention relates to (a) an antibody and/or pharmaceuticalcomposition of the present invention and (b) a vaccine for use as amedicament, for example for use in a method for the treatment of cancer.In one embodiment, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody and/orpharmaceutical composition of the present invention and (b) a vaccine.In one embodiment, the vaccine is a heat shock protein based tumorvaccine. In one embodiment, the vaccine is a heat shock protein basedpathogen vaccine.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In certain embodiments, HSPPCs used in methods disclosedherein comprise one or more heat shock proteins from the hsp60, hsp70,or hsp90 family of stress proteins complexed with antigenic peptides. Incertain embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, or combinations of two or more thereof.

In a specific embodiment, the heat shock protein peptide complex (HSPPC)comprises recombinant heat shock proteins (e.g., hsp70 or hsc70) or apeptide-binding domain thereof complexed with recombinant antigenicpeptides. Recombinant heat shock proteins can be produced by recombinantDNA technology, for example, using human hsc70 sequence as described inDworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) andGenBank accession no. P11142 and/or Y00371, each of which isincorporated herein by reference in its entirety. In certainembodiments, Hsp70 sequences are as described in Hunt and Morimoto Proc.Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accessionno. PODMV8 and/or M11717, each of which is incorporated herein byreference in its entirety. Antigenic peptides can also be prepared byrecombinant DNA methods known in the art.

In certain embodiments, the antigenic peptides comprise a modified aminoacid. In certain embodiments, the modified amino acid comprises apost-translational modification. In certain embodiments, the modifiedamino acid comprises a mimetic of a post-translational modification. Incertain embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg,Lys, or His that has been phosphorylated on a side chain hydroxyl oramine. In certain embodiments, the modified amino acid is a mimetic of aTyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylatedon a side chain hydroxyl or amine.

In a specific embodiment, an anti-LAG-3 (e.g., human LAG-3) antibodydisclosed herein is administered to a subject in combination with a heatshock protein peptide complex (HSPPC), e.g., heat shock protein peptidecomplex-96 (HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heatshock protein (Hsp), gp96, complexed to antigenic peptides. HSPPC-96 isa cancer immunotherapy manufactured from a subject's tumor and containsthe cancer's antigenic “fingerprint.” In certain embodiments, thisfingerprint contains unique antigens that are present only in thatparticular subject's specific cancer cells and injection of the vaccineis intended to stimulate the subject's immune system to recognize andattack any cells with the specific cancer fingerprint. Therefore, in oneembodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention in combination witha heat shock protein peptide complex (HSPPC) for use as a medicamentand/or for use in a method for the treatment of cancer.

In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from a tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In certainembodiments, the tumor tissue is non-necrotic tumor tissue. In certainembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In certain embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In some embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In certain embodiments, a subject is administered6-12 doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, all of which are herein incorporated by referencein their entireties: U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095,6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, an anti-LAG-3 antibody disclosed herein isadministered to a subject in combination with an adjuvant. Variousadjuvants can be used depending on the treatment context. Non-limitingexamples of appropriate adjuvants include, but not limited to, CompleteFreund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), montanideISA (incomplete Seppic adjuvant), the Ribi adjuvant system (RAS), TiterMax, muramyl peptides, Syntex Adjuvant Formulation (SAF), alum (aluminumhydroxide and/or aluminum phosphate), aluminum salt adjuvants, Gerbu®adjuvants, nitrocellulose absorbed antigen, encapsulated or entrappedantigen, 3 De-O-acylated monophosphoryl lipid A (3 D-MPL),immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands,mannan-binding lectin (MBL) ligands, STING agonists, immuno-stimulatingcomplexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX, and others.Other adjuvants include CpG oligonucleotides and double stranded RNAmolecules, such as poly(A) and poly(U). Combinations of the aboveadjuvants may also be used. See, e.g., U.S. Pat. Nos. 6,645,495;7,029,678; and 7,858,589, all of which are incorporated herein byreference in their entireties. In one embodiment, the adjuvant usedherein is QS-21 STIMULON.

In certain embodiments, an anti-LAG-3 antibody disclosed herein isadministered to a subject in combination with an additional therapeuticagent comprising a TCR. In certain embodiments, the additionaltherapeutic agent is a soluble TCR. In certain embodiments, theadditional therapeutic agent is a cell expressing a TCR. Therefore, inone embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention in combination withan additional therapeutic agent comprising a TCR for use as a medicamentand/or for use in a method for the treatment of cancer.

In certain embodiments, an anti-LAG-3 antibody disclosed herein isadministered to a subject in combination with a cell expressing achimeric antigen receptor (CAR). In certain embodiments, the cell is a Tcell.

In certain embodiments, an anti-LAG-3 antibody disclosed herein isadministered to a subject in combination with a TCR mimic antibody. Incertain embodiments, the TCR mimic antibody is an antibody thatspecifically binds to a peptide-MHC complex. For non-limiting examplesof TCR mimic antibodies, see, e.g., U.S. Pat. No. 9,074,000 and U.S.Publication Nos. US 2009/0304679 A1 and US 2014/0134191 A1, all of whichare incorporated herein by reference in their entireties.

The anti-LAG-3 (e.g., human LAG-3) antibody and the additionaltherapeutic agent (e.g., chemotherapeutic, radiotherapeutic, checkpointtargeting agent, IDO inhibitor, vaccine, adjuvant, a soluble TCR, a cellexpressing a TCR, a cell expressing a chimeric antigen receptor, and/ora TCR mimic antibody) can be administered separately, sequentially orconcurrently as separate dosage forms. In one embodiment, an anti-LAG-3(e.g., human LAG-3) antibody is administered parenterally, and an IDOinhibitor is administered orally.

An antibody or pharmaceutical composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intratumoral, conjunctival, intra-arterial, andsubcutaneous routes. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent for use as a spray. In certain embodiments, theantibody or pharmaceutical composition described herein is deliveredsubcutaneously or intravenously. In certain embodiments, the antibody orpharmaceutical composition described herein is delivered intratumorally.In certain embodiments, the antibody or pharmaceutical compositiondescribed herein is delivered intra-arterially. In certain embodiments,the antibody or pharmaceutical composition described herein is deliveredinto a tumor draining lymph node. In certain embodiments, the antibodyor pharmaceutical composition described herein is deliveredintranasally.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

An anti-LAG-3 (e.g., human LAG-3) antibody described herein can also beused to assay LAG-3 (e.g., human LAG-3) protein levels in a biologicalsample using classical immunohistological methods known to those ofskill in the art, including immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, or Western blotting.Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I,¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In), andtechnetium (⁹⁹Tc); luminescent labels, such as luminol; and fluorescentlabels, such as fluorescein and rhodamine, and biotin. Such labels canbe used to label an antibody described herein. Alternatively, a secondantibody that recognizes an anti-LAG-3 (e.g., human LAG-3) antibodydescribed herein can be labeled and used in combination with ananti-LAG-3 (e.g., human LAG-3) antibody to detect LAG-3 (e.g., humanLAG-3) protein levels. Therefore, in one embodiment, the presentinvention relates to the use of an antibody of the present invention forin vitro detection of LAG-3 (e.g., human LAG-3) protein in a biologicalsample. In a further embodiment, the present invention relates to theuse of an anti-LAG-3 antibody of the invention, for assaying and/ordetecting LAG-3 (e.g., human LAG-3) protein levels in a biologicalsample in vitro, optionally wherein the anti-LAG-3 antibody isconjugated to a radionuclide or detectable label, and/or carries a labeldescribed herein, and/or wherein an immunohistological method is used.

Assaying for the expression level of LAG-3 (e.g., human LAG-3) proteinis intended to include qualitatively or quantitatively measuring orestimating the level of LAG-3 (e.g., human LAG-3) protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level) or relatively (e.g., by comparing to the diseaseassociated protein level in a second biological sample). LAG-3 (e.g.,human LAG-3) polypeptide expression level in the first biological samplecan be measured or estimated and compared to a standard LAG-3 (e.g.,human LAG-3) protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” LAG-3 (e.g., human LAG-3) polypeptide level is known, it canbe used repeatedly as a standard for comparison. Therefore, in a furtherembodiment, the present invention relates to an in vitro method forassaying and/or detecting LAG-3 protein levels, for example human LAG-3protein levels, in a biological sample, comprising qualitatively orquantitatively measuring or estimating the level of LAG-3 protein, forexample of human LAG-3 protein, in a biological sample, by animmunohistological method.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing LAG-3 (e.g., human LAG-3). Methods forobtaining tissue biopsies and body fluids from animals (e.g., humans)are well known in the art. Biological samples include peripheralmononuclear blood cells.

An anti-LAG-3 (e.g., human LAG-3) antibody described herein can be usedfor prognostic, diagnostic, monitoring and screening applications,including in vitro and in vivo applications well known and standard tothe skilled artisan and based on the present description. Prognostic,diagnostic, monitoring and screening assays and kits for in vitroassessment and evaluation of immune system status and/or immune responsemay be utilized to predict, diagnose and monitor to evaluate patientsamples including those known to have or suspected of having an immunesystem-dysfunction or with regard to an anticipated or desired immunesystem response, antigen response or vaccine response. The assessmentand evaluation of immune system status and/or immune response is alsouseful in determining the suitability of a patient for a clinical trialof a drug or for the administration of a particular chemotherapeuticagent, a radiotherapeutic agent, or an antibody, including combinationsthereof, versus a different agent or antibody. This type of prognosticand diagnostic monitoring and assessment is already in practiceutilizing antibodies against the HER2 protein in breast cancer(HercepTest™, Dako) where the assay is also used to evaluate patientsfor antibody therapy using Herceptin®. In vivo applications includedirected cell therapy and immune system modulation and radio imaging ofimmune responses. Therefore, in one embodiment, the present inventionrelates to an anti-LAG-3 antibody and/or pharmaceutical composition ofthe present invention for use as a diagnostic. In one embodiment, thepresent invention relates to an anti-LAG-3 antibody and/orpharmaceutical composition of the present invention for use in a methodfor the prediction, diagnosis and/or monitoring of a subject having orsuspected to have an immune system-dysfunction and/or with regard to ananticipated or desired immune system response, antigen response orvaccine response. In another embodiment, the present invention relatesto the use of anti-LAG-3 antibody of the invention, for predicting,diagnosing and/or monitoring of a subject having or suspected to have animmune system-dysfunction and/or with regard to an anticipated ordesired immune system response, antigen response or vaccine response byassaying and/or detecting human LAG-3 protein levels in a biologicalsample of the subject in vitro.

In one embodiment, an anti-LAG-3 (e.g., human LAG-3) antibody can beused in immunohistochemistry of biopsy samples. In another embodiment,an anti-LAG-3 (e.g., human LAG-3) antibody can be used to detect levelsof LAG-3 (e.g., human LAG-3), or levels of cells which contain LAG-3(e.g., human LAG-3) on their membrane surface, which levels can then belinked to certain disease symptoms. Anti-LAG-3 (e.g., human LAG-3)antibodies described herein may carry a detectable or functional label.When fluorescence labels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-LAG-3 (e.g., humanLAG-3) antibodies described herein may carry a fluorescence label.Exemplary fluorescence labels include, for example, reactive andconjugated probes e.g., Aminocoumarin, Fluorescein and Texas red, AlexaFluor dyes, Cy dyes and DyLight dyes. An anti-LAG-3 (e.g., human LAG-3)antibody may carry a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu,¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Whenradioactive labels are used, currently available counting proceduresknown in the art may be utilized to identify and quantitate the specificbinding of anti-LAG-3 (e.g., human LAG-3) antibody to LAG-3 (e.g., humanLAG-3). In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques as known in the art. This can be achieved by contacting asample or a control sample with an anti-LAG-3 (e.g., human LAG-3)antibody under conditions that allow for the formation of a complexbetween the antibody and LAG-3 (e.g., human LAG-3). Any complexes formedbetween the antibody and LAG-3 (e.g., human LAG-3) are detected andcompared in the sample and the control. In light of the specific bindingof the antibodies described herein for LAG-3 (e.g., human LAG-3), theantibodies can be used to specifically detect LAG-3 (e.g., human LAG-3)expression on the surface of cells. The antibodies described herein canalso be used to purify LAG-3 (e.g., human LAG-3) via immunoaffinitypurification. Also included herein is an assay system which may beprepared in the form of a test kit for the quantitative analysis of theextent of the presence of, for instance, LAG-3 (e.g., human LAG-3) orLAG-3 (e.g., human LAG-3)/LAG-3 (e.g., human LAG-3) ligand complexes.The system or test kit, kit, or kit-of-parts may comprise a labeledcomponent, e.g., a labeled antibody, and one or more additionalimmunochemical reagents.

6.5 Polynucleotides, Vectors and Methods of Producing Anti-LAG-3Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a light chain variable region and/or heavy chain variableregion) that specifically binds to a LAG-3 (e.g., human LAG-3) antigen,and vectors, e.g., vectors comprising such polynucleotides forrecombinant expression in host cells (e.g., E. coli and mammaliancells). Provided herein are polynucleotides comprising nucleotidesequences encoding a heavy and/or light chain of any of the antibodiesprovided herein, as well as vectors comprising such polynucleotidesequences, e.g., expression vectors for their efficient expression inhost cells, e.g., mammalian cells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which specifically bind to aLAG-3 (e.g., human LAG-3) polypeptide and comprises an amino acidsequence as described herein, as well as antibodies which compete withsuch antibodies for binding to a LAG-3 (e.g., human LAG-3) polypeptide(e.g., in a dose-dependent manner), or which binds to the same epitopeas that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Tables 1, 3, 5, 6, and 7) ornucleotide sequences encoding a heavy chain comprising the VH FRs andCDRs of antibodies described herein (see, e.g., Tables 1, 2, 4, 6, and7).

Also provided herein are polynucleotides encoding an anti-LAG-3 (e.g.,human LAG-3) antibody that are optimized, e.g., by codon/RNAoptimization, replacement with heterologous signal sequences, andelimination of mRNA instability elements. Methods to generate optimizednucleic acids encoding an anti-LAG-3 (e.g., human LAG-3) antibody or afragment thereof (e.g., light chain, heavy chain, VH domain, or VLdomain) for recombinant expression by introducing codon changes and/oreliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly,all of which are herein incorporated by reference in their entireties.For example, potential splice sites and instability elements (e.g., A/Tor A/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In some embodiments, it can be desirable to alterone or more codons to encode a conservative mutation, e.g., a similaramino acid with similar chemical structure and properties and/orfunction as the original amino acid. Such methods can increaseexpression of an anti-LAG-3 (e.g., human LAG-3) antibody or fragmentthereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold or more relative to the expression of an anti-LAG-3 (e.g.,human LAG-3) antibody encoded by polynucleotides that have not beenoptimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-LAG-3 (e.g., human LAG-3) antibody described herein or a fragmentthereof (e.g., VL domain and/or VH domain) can hybridize to an antisense(e.g., complementary) polynucleotide of an unoptimized polynucleotidesequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody describedherein or a fragment thereof (e.g., VL domain and/or VH domain). Inspecific embodiments, an optimized nucleotide sequence encoding ananti-LAG-3 (e.g., human LAG-3) antibody described herein or a fragmenthybridizes under high stringency conditions to antisense polynucleotideof an unoptimized polynucleotide sequence encoding an anti-LAG-3 (e.g.,human LAG-3) antibody described herein or a fragment thereof. In aspecific embodiment, an optimized nucleotide sequence encoding ananti-LAG-3 (e.g., human LAG-3) antibody described herein or a fragmentthereof hybridizes under high stringency, intermediate or lowerstringency hybridization conditions to an antisense polynucleotide of anunoptimized nucleotide sequence encoding an anti-LAG-3 (e.g., humanLAG-3) antibody described herein or a fragment thereof. Informationregarding hybridization conditions has been described, see, e.g., U.S.Patent Application Publication No. US 2005/0048549 (e.g., paragraphs72-73), which is herein incorporated by reference in its entirety.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Tables 1, 6, and 7, and modified versions of theseantibodies can be determined using methods well known in the art, i.e.,nucleotide codons known to encode particular amino acids are assembledin such a way to generate a nucleic acid that encodes the antibody. Sucha polynucleotide encoding the antibody can be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier G et al.,(1994), BioTechniques 17: 242-6, which is herein incorporated byreference in its entirety), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-LAG-3 (e.g., human LAG-3) antibodies described hereincan be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theanti-LAG-3 (e.g., human LAG-3) antibodies). Hybridoma cells can serve asa source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells(e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof anti-LAG-3 (e.g., human LAG-3) antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable region, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in itsentirety.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein whichspecifically bind to LAG-3 (e.g., human LAG-3) and relatedpolynucleotides and expression vectors. Provided herein are vectors(e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-LAG-3 (e.g., human LAG-3) antibodiesor a fragment for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are host cells comprising suchvectors for recombinantly expressing anti-LAG-3 (e.g., human LAG-3)antibodies described herein (e.g., human or humanized antibody). In aparticular aspect, provided herein are methods for producing an antibodydescribed herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to LAG-3(e.g., human LAG-3) involves construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule, heavy and/or light chainof an antibody, or a fragment thereof (e.g., heavy and/or light chainvariable regions) described herein has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variableregion of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464, which are hereinincorporated by reference in their entireties) and variable regions ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein, or a fragment thereof, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein, or a fragment thereof. In otherembodiments, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable regionof an antibody described herein, or a fragment thereof, and a secondhost cell comprises a second vector comprising a polynucleotide encodinga light chain or a light chain variable region of an antibody describedherein. In specific embodiments, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-LAG-3 (e.g.,human LAG-3) antibody described herein. In certain embodiments, providedherein is a population of host cells comprising such first host cell andsuch second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-LAG-3 (e.g., human LAG-3)antibody described herein, and a second vector comprising apolynucleotide encoding a heavy chain/heavy chain variable region of ananti-LAG-3 (e.g., human LAG-3) antibody described herein.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715,which is herein incorporated by reference in its entirety). Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody moleculedescribed herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described hereinare CHO cells, for example CHO cells from the CHO GS System™ (Lonza). Incertain embodiments, the heavy chain and/or light chain of an antibodyproduced by a CHO cell may have an N-terminal glutamine or glutamateresidue replaced by pyroglutamate. In a particular embodiment, cells forexpressing antibodies described herein are human cells, e.g., human celllines. In a specific embodiment, a mammalian expression vector ispOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells suchas Escherichia coli, or eukaryotic cells (e.g., mammalian cells),especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking M K & Hofstetter H (1986) Gene 45:101-5; and Cockett M I et al., (1990) Biotechnology 8(7): 662-7, each ofwhich is herein incorporated by reference in its entirety). In certainembodiments, antibodies described herein are produced by CHO cells orNSO cells. In a specific embodiment, the expression of nucleotidesequences encoding antibodies described herein which specifically bindLAG-3 (e.g., human LAG-3) is regulated by a constitutive promoter,inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like, all of which are hereinincorporated by reference in their entireties. For example, pGEX vectorscan also be used to express foreign polypeptides as fusion proteins withglutathione 5-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to matrix glutathione agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region Elor E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated byreference in its entirety). Specific initiation signals can also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544, which is herein incorporated by reference in itsentirety).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-LAG-3 (e.g., human LAG-3)antibodies described herein are produced in mammalian cells, such as CHOcells.

In a specific embodiment, the antibodies described herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-LAG-3 (e.g., human LAG-3) antibody described herein canbe engineered. In specific embodiments, a cell provided herein stablyexpresses a light chain/light chain variable region and a heavychain/heavy chain variable region which associate to form an antibodydescribed herein.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-LAG-3 (e.g., humanLAG-3) antibody described herein or a fragment thereof. Such engineeredcell lines can be particularly useful in screening and evaluation ofcompositions that interact directly or indirectly with the antibodymolecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes in tk-, hgprt- or aprt-cells, respectively, all of which areherein incorporated by reference in their entireties. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56), all ofwhich are herein incorporated by reference in their entireties. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987), which is herein incorporated by reference in its entirety).When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse G F et al., (1983) Mol Cell Biol 3: 257-66,which is herein incorporated by reference in its entirety).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, each of which isherein incorporated by reference in its entirety). The coding sequencesfor the heavy and light chains can comprise cDNA or genomic DNA. Theexpression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotidesequences. For example, a bicistronic nucleic acid construct cancomprise in the following order a promoter, a first gene (e.g., heavychain of an antibody described herein), and a second gene and (e.g.,light chain of an antibody described herein). In such an expressionvector, the transcription of both genes can be driven by the promoter,whereas the translation of the mRNA from the first gene can be by acap-dependent scanning mechanism and the translation of the mRNA fromthe second gene can be by a cap-independent mechanism, e.g., by an IRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibodyor other different versions of an antibody (e.g., antibody fragments).When the antibody is recombinantly produced, it is also generallysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of theprotein preparation. When the antibody is produced by chemicalsynthesis, it is generally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a specific embodiment,antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to LAG-3 (e.g.,human LAG-3) can be produced by any method known in the art for thesynthesis of antibodies, for example, by chemical synthesis or byrecombinant expression techniques. The methods described herein employs,unless otherwise indicated, conventional techniques in molecularbiology, microbiology, genetic analysis, recombinant DNA, organicchemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel F M et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press, all of which areherein incorporated by reference in their entireties.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody whichspecifically binds to LAG-3 (e.g., human LAG-3) comprising culturing acell or host cell described herein. In one embodiment, the method isperformed in vitro. In a certain aspect, provided herein is a method ofmaking an antibody which specifically binds to LAG-3 (e.g., human LAG-3)comprising expressing (e.g., recombinantly expressing) the antibodyusing a cell or host cell described herein (e.g., a cell or a host cellcomprising polynucleotides encoding an antibody described herein). In aparticular embodiment, the cell is an isolated cell. In a particularembodiment, the exogenous polynucleotides have been introduced into thecell. In a particular embodiment, the method further comprises the stepof purifying the antibody obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York, whichis herein incorporated by reference in its entirety).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which isherein incorporated by reference in its entirety. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein or a fragment thereof, for example, light chain and/orheavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to LAG-3 (e.g., human LAG-3) as determined, e.g., by ELISA orother antigen-binding or competitive binding assay known in the art orin the examples provided herein. In particular embodiments, a monoclonalantibody can be a chimeric antibody or a humanized antibody. In certainembodiments, a monoclonal antibody is a monovalent antibody ormultivalent (e.g., bivalent) antibody. In particular embodiments, amonoclonal antibody is a monospecific or multispecific antibody (e.g.,bispecific antibody). Monoclonal antibodies described herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495, which is herein incorporated byreference in its entirety, or can, e.g., be isolated from phagelibraries using the techniques as described herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are well known in the art (see, forexample, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel F M et al., supra).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., LAG-3(e.g., human LAG-3)) used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986), herein incorporated byreference in its entirety). Additionally, a RIMMS (repetitiveimmunization multiple sites) technique can be used to immunize an animal(Kilpatrick K E et al., (1997) Hybridoma 16:381-9, herein incorporatedby reference in its entirety).

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., LAG-3 (e.g., human LAG-3)) and once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution. Incertain embodiments, lymph nodes of the immunized mice are harvested andfused with NSO myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asNSO cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X₆₃-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), eachof which is herein incorporated by reference in its entirety).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against LAG-3 (e.g., humanLAG-3). The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (MA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include antibody fragments which recognizespecific LAG-3 (e.g., human LAG-3) and can be generated by any techniqueknown to those of skill in the art. For example, Fab and F(ab′)2fragments described herein can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). A Fab fragmentcorresponds to one of the two identical arms of an antibody molecule andcontains the complete light chain paired with the VH and CH1 domains ofthe heavy chain. A F(ab′)2 fragment contains the two antigen-bindingarms of an antibody molecule linked by disulfide bonds in the hingeregion.

Further, the antibodies described herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with a scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108, all of which are herein incorporatedby reference in their entireties.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415, all of which are herein incorporated byreference in their entireties.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₃ and IgG₄. Humanized antibodies can be produced using a variety oftechniques known in the art, including but not limited to, CDR-grafting(European Patent No. EP 239400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089),veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596;Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G M et al.,(1994) Prot Engineering 7(6): 805-814; and Roguska M A et al., (1994)PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73, all of which are herein incorporated by referencein their entireties. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713, allof which are herein incorporated by reference in their entireties.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of whichare herein incorporated by reference in their entireties.

Further, antibodies that specifically bind to a LAG-3 (e.g., humanLAG-3) antigen can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” an antigen using techniques well known to thoseskilled in the art. See, e.g., Greenspan N S & Bona C A (1989) FASEB J7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438, eachof which is herein incorporated by reference in its entirety.

In particular embodiments, an antibody described herein, which binds tothe same epitope of LAG-3 (e.g., human LAG-3) as an anti-LAG-3 (e.g.,human LAG-3) antibody described herein, is a human antibody. Inparticular embodiments, an antibody described herein, whichcompetitively blocks (e.g., in a dose-dependent manner) any one of theantibodies described herein, from binding to LAG-3 (e.g., human LAG-3),is a human antibody. Human antibodies can be produced using any methodknown in the art. For example, transgenic mice which are incapable ofexpressing functional endogenous immunoglobulins, but which can expresshuman immunoglobulin genes, can be used. In particular, the human heavyand light chain immunoglobulin gene complexes can be introduced randomlyor by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of an antigen (e.g., LAG-3 (e.g., human LAG-3)). Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93,herein incorporated by reference in its entirety. For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096 and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598, all of which are hereinincorporated by reference in their entireties. Examples of mice capableof producing human antibodies include the Xenomouse™ (Abgenix, Inc.;U.S. Pat. Nos. 6,075,181 and 6,150,184), the HuAb-Mouse™ (Mederex,Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), all of whichare herein incorporated by reference in their entireties.

Human antibodies which specifically bind to LAG-3 (e.g., human LAG-3)can be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,4,716,111, and 5,885,793; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, all of which are herein incorporated byreference in their entireties.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that specifically bind to a targetantigen (e.g., LAG-3 (e.g., human LAG-3)). Such methods are known andare described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31, each of which is herein incorporated by reference in itsentirety.

6.6 Kits

Also provided, are kits comprising one or more antibodies describedherein, or pharmaceutical composition or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein. In some embodiments, the kitscontain a pharmaceutical composition described herein and anyprophylactic or therapeutic agent, such as those described herein. Incertain embodiments, the kits may contain a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits described herein contain a substantially isolated LAG-3 (e.g.,human LAG-3) antigen as a control. In another specific embodiment, thekits described herein further comprise a control antibody which does notreact with a LAG-3 (e.g., human LAG-3) antigen. In another specificembodiment, kits described herein contain one or more elements fordetecting the binding of an antibody to a LAG-3 (e.g., human LAG-3)antigen (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody can be conjugated to a detectablesubstrate). In specific embodiments, a kit provided herein can include arecombinantly produced or chemically synthesized LAG-3 (e.g., humanLAG-3) antigen. The LAG-3 (e.g., human LAG-3) antigen provided in thekit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which a LAG-3 (e.g., human LAG-3) antigen is attached.Such a kit can also include a non-attached reporter-labeled anti-humanantibody or anti-mouse/rat antibody. In this embodiment, binding of theantibody to the LAG-3 (e.g., human LAG-3) antigen can be detected bybinding of the said reporter-labeled antibody. In one embodiment, thepresent invention relates to the use of a kit of the present inventionfor in vitro assaying and/or detecting LAG-3 antigen (e.g., human LAG-3)in a biological sample.

7. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

7.1 Example 1: Generation and Characterization of Novel AntibodiesAgainst Human LAG-3

This example describes the generation and characterization of antibodiesthat bind to human Lymphocyte activation gene 3 (LAG-3), also known asCD223. In particular, this example describes the generation of murineantibodies that specifically bind to human LAG-3 and inhibit thefunction of human LAG-3.

7.1.1 Generation of Anti-LAG-3 Antibodies

Anti-LAG-3 antibodies were identified by generation and selection of animmunized Fab phage display library. First, total RNA was purified fromsingle-cell suspension of splenocytes from three individual micepreviously immunized with recombinant human LAG-3-Fc protein (R&DSystems, Cat #2319-L3-050) and recombinant cynomolgus monkey LAG-3-Fcprotein (Evitria, Custom order). Per mouse, a Fab library was thengenerated by random-primed cDNA using total RNA as a template to amplifyvariable regions from mouse antibody genes. Heavy and kappa chainamplicons were combined and cloned into phagemid vectors. Three roundsof selection were performed against recombinant LAG-3 proteins (humanLAG-3-Fc, human LAG-3-6His, and/or a 30 amino acid human LAG-3 peptide)and/or cells expressing cynomolgus LAG-3 to identify LAG-3-specific Fabphage clones. The 30 amino acid human LAG-3 peptide is a biotinylatedpeptide comprising the amino acid sequence ofGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 199). Periplasmic extracts ofselected Fab clones were then screened by ELISA or flow cytometry.Antibody sequencing and off-rate analysis were then performed onanti-LAG-3 Fabs.

A set of murine antibodies that bind to human LAG-3 were identified anddesignated as P01A12, P01C09, PO5E01, P05E03, P13A04, P13A06, P13B01,P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05, P13E02,P13F01, P13F02, P13F06, P13F09, P13G01, P13G04, P13G05, P13H05, P14A04,P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15CO2, P15E06,P15F06, P15G05, P16D04, and P16H05. The sequence information of thevariable regions of these antibodies is summarized in Table 6.

7.1.2 Binding of Anti-LAG-3 Fabs to LAG-3-Expressing Cells

The anti-LAG-3 Fabs from periplasmic extracts were tested for binding toLAG-3-expressing cells using flow cytometry. Briefly, wild type andhuman LAG-3-expressing Jurkat cells were plated at 2×10⁵ cells/well insample buffer (PBS (Gibco, Cat #10010-015)+0.5% FBS (Gibco, Cat#10270-106)) in 96-well U-bottom plates (Sarstedt). 22 μl of anti-LAG-3Fab periplasmic extract and 83 μl of diluted anti-c-myc antibody(Gentaur, Clone #9E10, Cat #04-cmyc-9E10) were incubated for 30 minutesat room temperature. 100 μl of the periplasmic extract/anti-c-mycmixture was added to cells and then incubated for one hour on ice. Cellswere washed three times and then incubated with a goat anti-mouse APC(BD Biosciences, Cat #550826) for 30 minutes on ice. Cells were washedthree times and then analyzed with a FACS machine (BD Accuri6).

As shown in FIGS. 1A-1C, all the anti-LAG-3 Fabs tested exhibitedbinding to Jurkat cells expressing human LAG-3. The apparent low levelof binding of PO5E01 was possibly due to low Fab concentration in theperiplasmic extracts. The binding of PO5E01 were confirmed in laterexperiments (data not shown).

7.1.3 Ligand Blocking Activity of Anti-LAG-3 Fabs or Antibodies

Anti-LAG-3 Fabs were tested for their ability to block the binding ofrecombinant human LAG-3 to MHC class II expressing cells. LAG-3-6His(Acro Biosystems, Cat #LA3-H5222) was pre-incubated at 10 μg/ml withanti-His biotin (Genscript, Clone #A00186, Cat #A00613) at 6 μg/ml for10 minutes at 4° C. This mixture was then incubated with either 10000ng/ml (FIG. 2A) or a serial dilution (21170, 7056.6, 2352.2, 784.0,261.3, 87.1, 29.0, or 9.7 ng/ml) (FIG. 2B) of anti-LAG-3 Fab or anegative control Fab not specific for LAG-3 for 60 minutes at 4° C. andthen incubated with 50,000 Raji cells for another 60 minutes at 4° C.Cultures were washed twice with sample buffer (PBS+2% FBS+0.09% sodiumazide) and then incubated with streptavidin-PE (Biolegend, Cat #405204)in sample buffer for 30 minutes at 4° C. Cells were washed twice andthen analyzed with a FACS Fortessa cytometer (Becton Dickinson).

As shown in FIGS. 2A and 2B, all the anti-LAG-3 Fabs tested blocked thebinding of cross-linked recombinant LAG-3-6His to MHC class II positiveRaji cells.

The variable regions of selected Fabs were cloned into human heavy chain(Ch1, Ch2, and Ch3) and light chain (CL) constant regions and expressedas full-length IgG₁ chimeric antibodies and tested in a similar ligandblocking assay as described above at various concentrations (96360,48180, 24090, 12045, 6022, 3011, 1505, 753, 376, 188, or 94 ng/ml).

The full length chimeric anti-LAG-3 antibodies tested all blocked theinteraction between recombinant LAG-3 and MHC class II expressing Rajicells (FIG. 3).

7.1.4 Effect of Anti-LAG-3 Antibody on Human PBMCs Upon StaphylococcusEnterotoxin a (SEA) Stimulation

The functional activity of the chimeric anti-LAG-3 antibody P13B02 wastested using primary human peripheral blood mononuclear cells (PBMCs)stimulated with Staphylococcus Enterotoxin A (SEA). Briefly,cryopreserved human PBMCs (Research Blood Components) were plated at 10⁵cells/well in RPMI1640 supplemented with Normocin™ (Invivogen, Cat#ant-nr-1) and 10% heat-inactivated FBS (Thermo Fisher Scientific, Cat#26140079) in 96-well NUNCLON delta surface plates (NUNC™). Cells werecultured with 100 ng/ml SEA (Toxin Technologies, Cat #at101red) and 10μg/ml P13B02 or an isotype control antibody for 5 days at 37° C., 5%CO2, and 97% humidity. Clarified supernatant was collected and stored at−80° C. until analysis. IL-2 levels were determined using AlphaLISA(Perkin Elmer, Cat #AL221C).

As shown in FIG. 4, the anti-LAG-3 antibody P13B02 increased IL-2production in human PBMCs stimulated with the SEA superantigen.

7.2 Example 2: Generation and Characterization of Humanized AntibodiesAgainst Human LAG-3

This example describes the humanization of the murine antibody P13B02and the characterization of the humanized antibodies.

7.2.1 Humanization of Murine Antibody P13B02

Homology matching was used to choose human acceptor framework regions tograft the CDRs of the murine antibody P13B02. Databases, e.g., adatabase of germline variable genes from the immunoglobulin loci ofhuman and mouse (the IMGT database (the international ImMunoGeneTicsinformation System®; Lefranc M P et al., (1999) Nucleic Acids Res 27(1):209-12; Ruiz M et al., (2000) Nucleic Acids Res 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res 29(1): 207-9; Lefranc M P (2003) NucleicAcids Res 31(1): 307-10; Lefranc M P et al., (2005) Dev Compo Immunol29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics &Proteomics 6(4): 253-64) or the VBASE2 (Retter I et al., (2005) NucleicAcids Res 33, Database issue D671-D674) or the Kabat database (Johnson Get al., (2000) Nucleic Acids Res 28: 214-218)) or publications (e.g.,Kabat E A et al., (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242), all of which are herein incorporated byreference in their entireties, may be used to identify the humansubfamilies to which the murine heavy and light chain variable regionsbelong and determine the best-fit human germline framework to use as theacceptor molecule. Selection of heavy and light chain variable regionsequences within these subfamilies to be used as acceptor may be basedupon sequence homology and/or a match of structure of the CDR1 and CDR2regions to help preserve the appropriate relative presentation of thesix CDRs after grafting.

Searching of the IMGT database, downloaded from IMGT.org, using BioEditSequence Alignment Editor (Hall, T. A. 1999. BioEdit: a user-friendlybiological sequence alignment editor and analysis program for Windows95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98, herein incorporated byreference in its entirety) indicated good homology between the P13B02heavy chain variable region framework and members of the human heavychain variable region subgroup IGHV1. Highest homologies and identitiesof both CDR and framework sequences were observed for germlinesequences: IGHV1-46*01 (SEQ ID NO: 153) (62% identity; 61 amino acidresidues out of 98); IGHV1-69-2*01 (SEQ ID NO: 154) (63% identity;62/98); IGHV1-3*01 (SEQ ID NO: 155) (64% identity; 63/98); IGHV1-24*01(SEQ ID NO: 156) (61% identity; 60/98); IGHV1-2*01 (SEQ ID NO: 157) (60%identity; 59/98); IGHV1-45*01 (SEQ ID NO: 158) (59% identity; 58/98);and IGHV1-18*01 (SEQ ID NO: 159) (60% identity; 59/98).

Using the same approach, P13B02 light chain variable region sequenceshowed good homology to members of the human light chain variable regionkappa subgroups IGKV3 and IGKV1. Highest homologies and identities ofboth CDRs and framework sequences were observed for germline sequences:IGKV3-20*01 (SEQ ID NO: 160) (59% identity; 57 amino acid residues outof 96); IGKV3D-15*01 and IGKV3-15*01 (SEQ ID NO: 161) (58% identity;56/96); IGKV3D-20*01 (SEQ ID NO: 162) (59% identity; 57/96); IGKV3D-7*01(SEQ ID NO: 163) (58% identity; 56/96); IGKV1-9*01 (SEQ ID NO: 164) (63%identity; 61/96); and IGKV3-11*01 (SEQ ID NO: 165) (60% identity;58/96).

As a starting point to the humanization process, a CDR grafted versionof mouse P13B02 VH was generated using framework regions 1,2 and 3 ofhuman IGHV1-46*01 (SEQ ID NO: 153) and framework region 4 of humanIGHJ1*01 (SEQ ID NO: 200) as human framework acceptor. A number of backmutations were made at positions that may affect the conformation ofCDRs or inter-variable region packing and therefore may be structurallyimportant for maintaining the full activity of the antibody (H0-H4; SEQID NOs: 56-60 and 220, respectively) (FIG. 5A). Similarly, a CDR graftedversion of mouse P13B02 VL was generated using framework regions 1, 2,and 3 of human IGKV3-20*01 (SEQ ID NO: 160) and framework 4 of humanIGKJ1*01 (SEQ ID NO: 201) as human framework acceptor (L0; SEQ ID NO:73) (FIG. 5B). Back mutations were generated at various positions(L1-L4; SEQ ID NOs: 74-77, respectively) (FIG. 5B). The sequences of thehumanized VHs H0-H4 and the humanized VLs L0-L4 are provided in Table 1.Table 10 shows positions that are different between mouse and humanantibody framework regions and are subjected to back mutation in atleast one of the humanized VHs or VLs described above. Table 11 showspositions that are different between mouse and human antibody frameworkregions and are subjected to back mutation in H1_R98K_L4M.

TABLE 10 Summary of framework back mutations in at least one of thehumanized VHs or VLs. Heavy chain variable region Kabat position P13B02VH IGHV1-46*01 (SEQ ID NO: 153) H4 M L H5 K V H12 V K H23 T K H27 F YH28 N T H29 I F H30 K T H48 I M H69 I M H71 A R H75 S T H76 N S H80 L MH81 Q E H94 T R Light chain variable region Kabat position P13B02 VLIGKV3-20*01 (SEQ ID NO: 160) L3 L V L22 T S L36 F Y L43 S A L47 W L L58V I L70 S D L71 Y F

TABLE 11 Summary of framework back mutations in H1_R98K_L4M. Kabatposition P13B02 VH IGHV1-46*01 (SEQ ID NO: 153) H4 M L H27 F Y H28 N TH29 I F H30 K T H69 I M H71 A R H94 T R

In addition, the HCDR3 of P13B02 contains a “RYD” motif, which has beendemonstrated previously to mimic the integrin binding motif “RGD.” Inorder to test whether the “RYD” motif could be removed without impactingbinding to LAG-3, an amino acid substitution R98K or D100E, numberedaccording to Kabat definition, was introduced in the heavy chain.

A panel of 37 humanized antibodies, designated as P13B02-01 toP13B02-37, were designed based on the description above and generated asfull length IgG₁ antibodies. P13B02-30 were generated in three versions:a full length antibody containing IgG₁ G1m17 allotype, referred to asP13B02-30 (IgG₁); a full length antibody containing IgG₁ G1m17 allotypewith a N297A mutation, referred to as P13B02-30 (IgG₁ G1m17 N297A); anda full length antibody containing IgG₁ G1m3 allotype with a N297Amutation, referred to as P13B02-30 (IgG₁ G1m3 N297A). The sequenceinformation of the variable regions of P13B02-01 to P13B02-37 issummarized in Table 7. Two batches of P13B02-30 (IgG₁ G1m3 N297A) wereexamined for post-translational processing in the producer cells. Allthese 37 humanized antibodies retained binding to recombinant humanLAG-3 in surface plasmon resonance analysis (data not shown).

7.2.2 Binding of Humanized Anti-LAG-3 Antibodies to Human LAG-3

The anti-LAG-3 antibodies were tested for binding to primary human Tcells using flow cytometry. Cryopreserved human PBMCs (Research BloodComponents) were plated at 10⁶ cells/ml in RPMI1640 supplemented withNormocin™ (Invivogen, Cat #ant-nr-1) and 10% heat-inactivated FBS(Thermo Fisher Scientific, Cat #26140079) in a T-75 flask (Corning) inthe presence of 100 ng/ml SEA (Toxin Technologies, Cat #at101red) for 5days at 37° C., 5% CO2, and 97% humidity. Cultured PBMCs were thenplated at 10⁵ cells/well in 96-well U-bottom plates (Nunc). Cells wereincubated with 10 μg/ml of anti-LAG-3 antibody for 30 minutes on ice.Cells were washed three times and then incubated with anti-CD4-PE/Cy7(Biolegend, Clone #OKT4, Cat #317414), anti-CD8-FITC (Biolegend, Clone#RPA-T8, Cat #301060), anti-CD3-APC (BD Biosciences, Clone #SP34-2, Cat#557597), LIVE/DEAD® Fixable Near-IR Dead Cell Stain (Life Technologies,Cat #L10119), Fc block (BD Biosciences, Cat #422302), and goatanti-human IgG-PE (ThermoFisher, Cat #PA1-74408). Cells were incubatedfor 30 minutes on ice, washed, and analyzed with a FACS machine (BDCanto). As shown in FIG. 6A, the chimeric antibody P13B02 and all thehumanized antibodies tested exhibited binding to primary CD4+ human Tcells stimulated with the SEA superantigen.

Next, human PBMCs were activated using the SEA superantigen similarly asdescribed above and incubated with serially diluted (50000, 15000, 4500,1350, 407, 122, 37, or 11 ng/ml) P13B02-16 or an isotype controlantibody. Binding was analyzed with a FACS Fortessa cytometer (BectonDickinson). The humanized antibody P13B02-16 bound to activated primaryhuman CD4+ T cells in a dose-dependent manner (FIG. 6B).

7.2.3 Ligand Blocking Activity of Humanized Anti-LAG-3 Antibodies

Next, the ability of the humanized anti-LAG-3 antibodies to block theinteraction between cross-linked recombinant LAG-3-6His and MHC class IIexpressing Raji cells was examined as described above. The humanizedantibodies P13B02-06, P13B02-07, P13B02-16, P13B02-26, and P13B02-27,all of which include a human IgG₁ constant region, were tested at 57820,28910, 14455, 7228, 3613, 1807, 903, 452, 226, 113, and 56 ng/ml (FIG.7A). The humanized antibody P13B02-30 (IgG₁ G1m17 N297A) was tested at96360, 48180, 24090, 12045, 6022, 3011, 1505, 753, 376, 188, and 94ng/ml (FIG. 7B).

As shown in FIGS. 7A and 7B, all the humanized antibodies testedeffectively blocked LAG-3 binding to MHC class II positive Raji cells.

7.2.4 Effect of Humanized Anti-LAG-3 Antibody on Human PBMCs UponStaphylococcus Enterotoxin a (SEA) Stimulation

The functional activity of the humanized antibody P13B02-30 (IgG₁) wasassessed using primary human PBMCs stimulated by StaphylococcusEnterotoxin A (SEA). Cryopreserved human PBMCs (Research BloodComponents) were plated at 10⁵ cells/well in RPMI1640 supplemented withNormocin™ (Invivogen, Cat #ant-nr-1) and 10% heat-inactivated FBS(Thermo Fisher Scientific, Cat #26140079) in 96-well NUNCLON deltasurface plates (NUNC™). Cells were cultured with 100 ng/ml SEA (ToxinTechnologies, Cat #at101red) and 10 μg/ml P13B02-30 (IgG₁) or an isotypecontrol antibody for 5 days at 37° C., 5% CO2, and 97% humidity.Clarified supernatant was collected and stored at −80° C. untilanalysis. IL-2 levels were determined using AlphaLISA (Perkin Elmer, Cat#AL221C).

As shown in FIG. 8A, the anti-LAG-3 antibody P13B02-30 (IgG₁) enhancedIL-2 production in human PBMCs stimulated with the SEA superantigen.

In a similar experiment, cryopreserved human PBMCs were cultured with100 ng/ml SEA (Toxin Technologies, Cat #at101red) and 10 μg/ml P13B02-16(IgG₁) or an isotype control antibody in the presence or absence of 5μg/ml of anti-PD-1 antibody pembrolizumab (Pembro) (Myoderm), anti-PD-1antibody nivolumab (Nivo) (Myoderm), three different anti-PD-L1antibodies, or anti-CTLA-4 antibody ipilimumab (Ipi) (Myoderm) for 5days at 37° C., 5% CO2, and 97% humidity. Clarified supernatant wascollected and stored at −80° C. until analysis. IL-2 levels weredetermined using AlphaLISA (Perkin Elmer, Cat #AL221C). Anti-PD-L1antibody #1 was generated based on the variable region sequences ofantibody A09-246-2 provided in U.S. Application Publication No.US2014/0341917 (herein incorporated by reference in its entirety).Anti-PD-L1 antibody #2 was generated based on the variable regionsequences of antibody 2.14H9OPT provided in U.S. Pat. No. 8,779,108(herein incorporated by reference in its entirety). Anti-PD-L1 antibody#3 was generated based on sequences provided in U.S. Pat. No. 8,217,149(herein incorporated by reference in its entirety). The sequences ofthese three anti-PD-L1 antibodies are listed in Table 12.

TABLE 12 Sequences of anti-PD-Ll antibodies SEQ ID NO: DescriptionAmino acid sequence 202 anti-PD-LlEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLE antibody #1WVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTA  heavy chainVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 203 anti-PD-L1QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP antibody #1KLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSS  light chainYTSSSTRVFGTGTKVTVLQPKANPTVTLFPPSSEELQANKATLVCL ISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 204 anti-PD-L1EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLE antibody #2WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTA  heavy chainVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKS ISGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 205 anti-PD-L1EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPR antibody #2LLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY  light chainGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 206 anti-PD-L1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE antibody #3WVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA  heavy chainVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 207 anti-PD-L1DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL antibody #3LIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYL light chainYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

As shown in FIG. 8B, P13B02-16 (IgG₁), either alone or in combinationwith the anti-PD-1 antibody pembrolizumab or nivolumab, the anti-PD-L1antibody #1, #2, or #3, or the anti-CTLA-4 antibody ipilimumab, enhancedIL-2 production in human PBMCs in the presence of the SEA superantigen.

7.2.5 Effect of Humanized Anti-LAG-3 Antibody on Cytokine Production ofTumor Infiltrating Lymphocytes

The anti-LAG-3 antibody P13B02-30 (IgG₁ Glm3 N297A) was further assessedfor its ability to stimulate cytokine production of activated primarytumor infiltrating lymphocytes (TILs), alone or in combination with ananti-PD-1 antibody. Single-cell suspensions from fresh renal cellcarcinoma (RCC) (stage I) or colorectal cancer (CRC) (stage II) tumors(UMass Medical School, Worcester, Mass.) were isolated via mechanicalmicrodissection. In some cases, depending on the level of fibrosis,enzymatic digestion was necessary (Liberase and DNAseI, Roche). Cellswere rested at 5×10⁴ cells/well in RPMI1640 supplemented with Normocin™(Invivogen, Cat #ant-nr-1), recombinant human IL-2 (20 U/ml, R&DSystems, Cat #202-IL-010), and 10% heat-inactivated FBS (Thermo FisherScientific, Cat #26140079) in 96-well NUNCLON delta surface plates(NUNC™, Cat #143761) for 1 day. On the following day, the samples werecentrifuged and fresh culture media containing the antibodies ofinterest, P13B02-30 (IgG₁ Glm3 N297A) at 20 μg/ml and the anti-PD-1antibody pembrolizumab (Pembro) (Myoderm) at 5 μg/ml, and anti-CD3/CD28microbeads (1:1 bead:cell ratio), was added at a final volume of 100 μland allowed to incubate for 3 days at 37° C. and 5% CO2. Cell-freesupernatant was collected and stored at −80° C. until analysis. TNFαlevels were determined using AlphaLISA (Perkin Elmer, Cat #AL208C).

As shown in FIGS. 9A and 9B, the anti-LAG-3 antibody P13B02-30 (IgG₁Glm3 N297A) enhanced TNFα production of activated primary TILs from RCCor CRC tumors.

7.2.6 Anti-LAG-3 Antibody Enhances T Cell Activation in a LAG-3-MediatedCell Suppression Assay

In this example, an NFAT-luciferase reporter line was used to assess theinhibitory effect of the anti-LAG-3 antibody P13B02-30 (IgG₁ Glm3 N297A)against LAG-3 in a cell suppression assay. Two experiments wereperformed, as described below.

In each of the two experiments, Jurkat-NFAT-LAG-3 cells were suspendedto a 2.5×working concentration of 1×10⁶ cells/mL in assay medium(RPMI+10% heat-inactivated FBS+1% Pen/Strep). Raji cells were suspendedto a 3.33× working concentration of 1×10⁶ cells/mL in assay medium.Staphylococcal Enterotoxin E (Toxin Technology) was prepared at a 10×working concentration of 0.04 ng/mL in assay medium. A serial dilutionof anti-LAG-3 antibody or isotype antibody was prepared in assay medium.In the first experiment, the antibody concentrations ranged from 0.2-50μg/mL. In the second experiment, the antibody concentrations ranged from0.1-100 μg/mL). After the serial dilutions of the antibodies wereprepared, 40 μL of Jurkat-NFAT-LAG-3 cells and 20 μL of antibodysolution were pre-incubated for 30 minutes at 37° C. and 5% CO2 inU-bottom 96-well plates. 30 μL of Raji cells and 10 μL of StaphylococcalEnterotoxin E were added to the 96-well plates and incubated for 5-6hours. 100 μL of Bio-Glo Luciferase (Promega) was then added to eachwell and luminescence was recorded using EnVision Plate Reader (PerkinElmer) after 10-15 minutes.

As shown in FIGS. 10A and 10B, the anti-LAG-3 antibody significantlyincreased NFAT-luciferase reporter signal, relative to isotype controlantibody, in a dose-dependent manner.

7.3 Example 3: Epitope Mapping of Anti-LAG-3 Antibody

In this example, the epitope of the anti-LAG-3 antibody P13B02-30 wascharacterized as described below.

7.3.1 Epitope Mapping of Anti-LAG-3 Antibody Using Hydrogen-DeuteriumExchange (HDX) Mass Spectrometry

Anti-LAG-3 F(ab′)2 was generated from P13B02-30 (IgG₄ S228P) using theFraglT Kit (Genovis, Cat #A2-FR2-100). The interaction of anti-LAG-3F(ab′)2 with human LAG-3 was studied using hydrogen-deuterium exchange(HDX) mass spectrometry.

For pepsin/protease XIII digestion, 7.9 μg of recombinant his-taggedhuman LAG-3 (Sino Biological, Cat #16498-H08H) in 125 μl control buffer(50 mM phosphate, 100 mM sodium chloride, pH 7.4) was denatured byadding 125 μl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (finalpH 2.5) and incubating the mixture for 5 minutes at 20° C. The mixturewas then subjected to on-column pepsin/protease XIII digestion using anin-house packed pepsin/protease XIII (w/w, 1:1) column and the resultantpeptides were analyzed using a UPLC-MS system comprised of a WatersAcquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap MassSpectrometer (Thermo). The peptides were separated on a 50 mm×1 mm C8column with a 19-minute gradient from 2-30% solvent B (0.2% formic acidin acetonitrile). Peptide identification was conducted through searchingMS/MS data against the human LAG-3 sequence with Mascot. The masstolerance for the precursor and productions was 10 ppm and 0.05 Da,respectively.

20 μl human LAG-3 (7.9 μg) or 20 μl human LAG-3/F(ab′)2 mixture (7.9 μg:15.8 μg) was incubated with 105 μl deuterium oxide labeling buffer (50mM sodium phosphate, 100 mM sodium chloride, pD 7.4) for 0 second, 60seconds, 300 seconds, 1800 seconds, 7200 seconds, and 14400 seconds at20° C. Hydrogen/deuterium exchange was quenched by adding 125 μl of 4 Mguanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5).Subsequently, the quenched samples were subjected to on columnpepsin/protease XIII digestion and LC-MS analysis as described above.The mass spectra were recorded in MS only mode. Raw MS data wereprocessed using HDX WorkBench, software for the analysis of H/D exchangeMS data (J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521, hereinincorporated by reference in its entirety). The deuterium levels werecalculated using the average mass difference between the deuteratedpeptide and its native form (to).

The sequence coverage achieved for human LAG-3 was 96.6%. While most ofthe human LAG-3 peptides displayed identical or similar deuterium levelswith and without the anti-LAG-3 F(ab′)2, several peptide segments werefound to have significantly decreased deuterium incorporation uponF(ab′)2 binding. A strong decrease in deuterium uptake was observed at aregion consisting of the amino acid sequence of SEQ ID NO: 215(SPTIPLQDLSL). The residues are numbered according to SEQ ID NO: 166.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

What is claimed:
 1. An isolated antibody that specifically binds tohuman LAG-3, comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2, and CDRH3, whereinCDRH1, CDRH2, and CDRH3 comprise the amino acid sequences of SEQ ID NOs:79, 90, and 98, respectively; and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 73. 2. The isolatedantibody of claim 1, wherein the amino acid sequence of the light chainvariable region consists of the amino acid sequence of SEQ ID NO:
 73. 3.The isolated antibody of claim 1, wherein the antibody comprises a lightchain comprising the amino acid sequence of SEQ ID NO:
 187. 4. Theisolated antibody of claim 3, wherein the amino acid sequence of thelight chain consists of the amino acid sequence of SEQ ID NO:
 187. 5. Anisolated antibody that specifically binds to human LAG-3, comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 65; and a light chain variable region comprising complementaritydetermining regions CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences of SEQ ID NOs: 100, 104, and105, respectively.
 6. The isolated antibody of claim 5, wherein theamino acid sequence of the heavy chain variable region consists of theamino acid sequence of SEQ ID NO:
 65. 7. The isolated antibody of claim5, wherein the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO:
 169. 8. The isolated antibody of claim 7,wherein the amino acid sequence of the heavy chain consists of the aminoacid sequence of SEQ ID NO:
 169. 9. An isolated antibody thatspecifically binds to human LAG-3, comprising a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 65; and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:73.
 10. The isolated antibody of claim 9, wherein the amino acidsequence of the heavy chain variable region consists of the amino acidsequence of SEQ ID NO: 65; and the amino acid sequence of the lightchain variable region consists of the amino acid sequence of SEQ ID NO:73.
 11. The isolated antibody of claim 9, wherein the antibody comprisesa heavy chain comprising the amino acid sequence of SEQ ID NO:
 169. 12.The isolated antibody of claim 11, wherein the amino acid sequence ofthe heavy chain consists of the amino acid sequence of SEQ ID NO: 169.13. The isolated antibody of claim 9, wherein the antibody comprises alight chain comprising the amino acid sequence of SEQ ID NO:
 187. 14.The isolated antibody of claim 13, wherein the amino acid sequence ofthe light chain consists of the amino acid sequence of SEQ ID NO: 187.15. An isolated antibody that specifically binds to human LAG-3,comprising a heavy chain and a light chain, wherein the heavy chaincomprises the amino acid sequence of SEQ ID NO: 169; and the light chaincomprises the amino acid sequence of SEQ ID NO:
 187. 16. The isolatedantibody of claim 15, wherein the amino acid sequence of the heavy chainconsists of the amino acid sequence of SEQ ID NO:
 169. 17. The isolatedantibody of claim 15, wherein the amino acid sequence of the light chainconsists of the amino acid sequence of SEQ ID NO:
 187. 18. The isolatedantibody of claim 15, wherein the amino acid sequence of the heavy chainconsists of the amino acid sequence of SEQ ID NO: 169, and the aminoacid sequence of the light chain consists of the amino acid sequence ofSEQ ID NO:
 187. 19. A pharmaceutical composition comprising the antibodyof claim 1 and a pharmaceutically acceptable carrier or excipient.
 20. Apharmaceutical composition comprising the antibody of claim 2 and apharmaceutically acceptable carrier or excipient.
 21. A pharmaceuticalcomposition comprising the antibody of claim 3 and a pharmaceuticallyacceptable carrier or excipient.
 22. A pharmaceutical compositioncomprising the antibody of claim 4 and a pharmaceutically acceptablecarrier or excipient.
 23. A pharmaceutical composition comprising theantibody of claim 5 and a pharmaceutically acceptable carrier orexcipient.
 24. A pharmaceutical composition comprising the antibody ofclaim 6 and a pharmaceutically acceptable carrier or excipient.
 25. Apharmaceutical composition comprising the antibody of claim 7 and apharmaceutically acceptable carrier or excipient.
 26. A pharmaceuticalcomposition comprising the antibody of claim 8 and a pharmaceuticallyacceptable carrier or excipient.
 27. A pharmaceutical compositioncomprising the antibody of claim 9 and a pharmaceutically acceptablecarrier or excipient.
 28. A pharmaceutical composition comprising theantibody of claim 10 and a pharmaceutically acceptable carrier orexcipient.
 29. A pharmaceutical composition comprising the antibody ofclaim 15 and a pharmaceutically acceptable carrier or excipient.
 30. Apharmaceutical composition comprising the antibody of claim 18 and apharmaceutically acceptable carrier or excipient.
 31. A pharmaceuticalcomposition comprising the antibody of claim 11 and a pharmaceuticallyacceptable carrier or excipient.
 32. A pharmaceutical compositioncomprising the antibody of claim 12 and a pharmaceutically acceptablecarrier or excipient.
 33. A pharmaceutical composition comprising theantibody of claim 13 and a pharmaceutically acceptable carrier orexcipient.
 34. A pharmaceutical composition comprising the antibody ofclaim 14 and a pharmaceutically acceptable carrier or excipient.
 35. Apharmaceutical composition comprising the antibody of claim 16 and apharmaceutically acceptable carrier or excipient.
 36. A pharmaceuticalcomposition comprising the antibody of claim 17 and a pharmaceuticallyacceptable carrier or excipient.